Olea europaea cultivar, native in the Mediterranean basin, has expanded worldwide, mainly due to the olive oil industry. This expansion is attributed to the benefits of olive oil consumption, since this product is rich in nutritional and bioactive compounds. However, the olive industry generates high amounts of wastes, which could be related to polluting effects on soil and water. To minimize the environmental impact, different strategies of revalorization have been proposed. In this sense, the aim of this work was to develop high cosmetic value added oleuropein-enriched extracts (O20 and O30), a bioactive compound from olive byproducts, performing a comprehensive characterization using high performance liquid chromatography coupled to mass spectrometry and evaluate their bioactivity by in vitro assays. A total of 49 compounds were detected, with oleuropein and its derivatives widely found in O30 extract, whereas iridoids were mainly detected in O20 extract. Moreover, 10 compounds were detected for the first time in olive leaves. Both extracts demonstrated strong antioxidant and antiradical activities, although O30 showed higher values. In addition, radical oxygen and nitrogen species scavenging and enzyme inhibition values were higher in O30, with the exception of HOCl and hyaluronidase inhibition assays. Regarding cell viability, olive byproduct extracts did not lead to a decrease in keratinocytes viability until 100 µg/mL. All data reported by the present study reflect the potential of industrial byproducts as cosmetic ingredients. Olea europaea L.), native in the Mediterranean basin, have spread to many countries and adapted to different pedoclimatic conditions. This cultivar has expanded in Asia, America and Oceania mainly due to the olive oil industry, although the Mediterranean area is the most important region in terms of olives with almost 20 M tons of olive production in 2018 , ferric sulfate, fluorescein, Folin–Ciocalteu reagent, potassium persulfate, TPTZ  and Trolox  from Sigma-Aldrich . From Panreac , gallic acid, dehydrated sodium phosphate, trihydrated sodium acetate, sodium acetate, ferric chloride, hydrochloric acid and sodium carbonate were purchased. To measure Reactive Oxygen/Nitrogen Species (ROS/RNS) scavenging, dihydrorhodamine 123 (DHR), sodium hypochlorite solution with 4% available chlorine, β-nicotinamide adenine dinucleotide (NADH), phenazine methosulphate (PMS) and nitroblue tetrazolium chloride (NBT) were purchase from Sigma Aldrich . Dimethylsulfoxide (DMSO) was obtained from AppliChem .To measure hyaluronidase and elastase inhibitions, all reagents, hyaluronidase from bovine testes Type I–S, hyaluronic acid as substrate, sodium acetate/phosphate/chloride, bovine serum albumin, buffer HEPES, NaCl, human leucocyte elastase, substrate MeOSuc-Ala-Ala-Pro-Val-pNA and elastatinal, were purchase from Sigma .TM, Hank’s Balanced Salt Solution (HBSS), non-essential amino acids, penicillin, streptomycin and trypsin–EDTA were obtained from Invitrogen Corporation . Trypan blue dye was purchased from Gibco . Tissue culture flasks were acquired from Orange Scientific .For cell viability, human immortalized non-tumorigenic keratinocytes cell line HaCaT  was obtained from CLS Cell Lines Service, Germany. Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), GlutaMAXO. europaea fruits and leaves (O20 = 20% oleuropein and O30 = 30% oleuropein + 10% triterpenes) were obtained from Natac company . These extracts were obtained by different process. Briefly, O20 was attained by a first ethanol extraction followed by filtration and concentration steps. After that, a second water extraction was made, followed by filtration, thermal treatment, vacuum drying, milling and standardization steps. O30 was obtained through two extraction processes running in parallel with different filtration and drying methodologies. The two milled final extracts were homogenized and standardized. The commercial extracts were supplied as dry residue then filtered with a 0.2 µm sterile filter, evaporated and lyophilized according to the manufacturer’s procedure. Both extracts (O20 and O30) were dissolved in ethanol:water  at a final concentration of 1 mg/mL for further experiments, except for the HPLC analysis  and cell assays (extracts at 1 mg/mL were dissolved in the culture medium used); then, extracts were vortexed for 1 min, sonicated for 20 min, centrifuged for 5 min at 17,000× g in a Sorvall ST 16 R centrifuge , filtered through a 0.20 mm filter and kept at −20 °C under light-free conditions until further experiments. Preliminary studies were performed for each assay to investigate the influence of the solvents  in the composition of the extracts.Two different oleuropein-enriched extracts from The ACQUITY UPLC H-Class System  was used for the separation of the bioactive compounds from both extracts (5 mg/mL) at 22 °C with a Zorbax Eclipse Plus C18 column . The mobile phases were acetic acid 0.5% (solvent A) and methanol (solvent B). This multistep linear gradient was applied: 0 min, 100% A; 5 min, 75% A; 20 min 61% A; 30 min, 40% A; 38 min, 0% A; 46 min, 100% A. The initial conditions were maintained for 10 min. The injection volume was 10 μL. The flow rate used was set at 0.4 mL/min.−1, desolvation temperature = 500 °C, cone gas flow = 50 L h−1, source temperature = 100 °C, capillary voltage = 2.2 kV, cone voltage = 30 V and collision energy = 20 eV. Full-scan mode was used (m/z = 50–1200). Scan duration was 0.1 s, and resolution was 20,000 FWHM. The MS data were processed through the open-source software MZmine.The HPLC analysis was coupled to QTOF/MS . The determination of the compounds was carried out using an electrospray source operating in negative ionization mode under the following conditions: MS acquisition was performed using two parallel scan functions by rapid switching, in which one scan was operated at low collision energy in the gas cell (4 eV) and the other at an elevated collision energy (MSE energy linear ramp: from 20 to 60 eV); desolvation gas flow = 700 L hv/v) and 120 μL of 7.5% (w/v) Na2CO3 solution. The microplate was incubated at 45 °C for 15 min in a Synergy Mx Monochromator-Based Multimode Microplate reader  and then left to stand in the dark for 30 min, at room temperature. Then, the absorbance was measured at 765 nm, and TPC was calculated based on the calibration curves of gallic acid and expressed as mg of gallic acid equivalents (GAE)/g of dry extract. FRAP assay was performed according to the procedure described by Benzie and Strain [3 6H2O solution). Ferrous sulphate was employed as standard (12.5–200 μM). After incubating the microplate at 37 °C for 10 min, the absorbance was recorded at 593 nm for 4 min. Results were expressed as mmol of ferrous sulphate equivalents (FeSO4)/g dry extract. The TEAC was determined by ABTS radical scavenging capacity assay following the procedure described by Miller et al. [•+) working solution was prepared by adding ABTS•+ stock solution to 2.45 mM potassium persulfate, and the final mixture was kept in the dark at room temperature, for 12 to 24 h. Prior to use, the ABTS•+ working solution was diluted with ultrapure water until reaching an absorbance of 0.700 (±0.02) at 734 nm. In a nutshell, 300 μL of ABTS•+ solution and 30 μL of extracts (250 μg/mL) or standard were mixed for 45 s, and the absorbance was measured after 5 min at 734 nm. Trolox (0.5–30 μM) was used as standard to plot a calibration curve. TEAC results were expressed as mmol of Trolox equivalents/g dry extract. The ORAC assay was performed to assess the capacity of the extracts to scavenge peroxyl radicals following the methodology described by Ou et al. [2PO4 and 0.2 M Na2HPO4  at pH 7.4. The microplate was incubated at 37 °C, and the fluorescence was read at 520 nm (excitation wavelength at 485 nm) for 2 h. The results were expressed as mmol of Trolox equivalents/g dry extract. Three independent experiments were carried out for each assay.The Folin-Ciocalteu method was carried out to measure the total phenolic content (TPC) as firstly validated by Singleton and Rossi , with mid Strain , with slr et al.  with minu et al.  and modiu et al. . A 56 nM50. The IC50 values were automatically calculated by GraphPad Prism 7 software.The extracts and positive controls  were previously dissolved in phosphate buffer used for each procedure. These methods were performed in a Synergy HT Microplate Reader . Three independent experiments using six concentrations, in duplicate, were carried out for each assay. Results were calculated from the curves of inhibition percentage versus antioxidant concentration using the GraphPad Prism 7 software . Prior to the assays, the absorption of the extract was studied at the proper wavelengths for each method. The methods for superoxide radical scavenging activity, hypochlorous acid scavenging assay and nitric oxide scavenging assay were carried out following the reported literature . Resultsp-nitroaniline, which was hydrolyzed from the substrate, MeOSuc-Ala-Ala-Pro-Val-pNa, by elastase, was read with a maximum absorbance at 405 nm. In brief, MeOSuc-Ala-Ala-Pro-Val-pNa was prepared in buffer (pH 8.0), and this solution was added to the samples. The solutions were vortexed and pre-incubated at 37 °C for 10 min before an elastase solution was added. After that, they were incubated at 37 °C for 30 min, and the absorbance was measured at 405 nm. All measurements were made in triplicate.Hyaluronidase and elastase inhibitory assays were performed following the method describe by Nema et al., 2011 and 2013 ,32. BrieTM-I, 10% inactivated fetal calf serum, 100 U/mL penicillin, 100 mG/mL streptomycin and 0.25 mG/mL amphotericin B, in a 5% CO2 environment at 37 °C . The number of viable cells was periodically assessed by the trypan blue exclusion assay. Keratinocytes  were exposed to different concentrations (0.1–1000 µg/mL) of extract for 24 h at 37 °C and in a water saturated atmosphere with 5% CO2 using an incubator. Following the supplier instructions, the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay was performed to estimate the intestinal cells’ viability by the determination of the number of viable cells through a colorimetric reaction. Briefly, cells were straightly placed in 96-well microplate (density of 25 × 103 cells/well) and incubated at 37 °C for 24 h. Afterwards, cells were exposed to different concentrations of O20 and O30 extracts, positive control (1% (v/v) Triton X-100) and negative control (DMEM) for 24 h. After the supernatant removal, the MTT was added to each well, and the microplate was incubated at 37 °C for 3 h to promote the development of formazan crystals. Then, the MTT solution was removed, and the blue formazan crystals were eluted with DMSO. Absorbance was read at 570 nm with a background subtraction at 690 nm.The cell viability assay was carried out according to the method described by Lameirão et al. . HaCaT cpost hoc comparisons of the means were performed with Tukey’s HSD test. In all cases, a denoting significance was accepted for p < 0.05. Regarding ROS and RNS scavenging assays, at least three independent experiments using six concentrations, in duplicate, were performed. Results were determined from the curves of inhibition percentage versus antioxidant concentration using GraphPad Prism 7 software .All experiments were carried out at least in triplicate. The results were presented as mean ± standard deviation of at least three independent experiments. IBM SPSS Statistics 24.0 software  was used for the statistical analyses of data. The differences between samples were investigated by one-way ANOVA, and m/z, molecular formula, MS/MS fragments and the presence of the compound in each extract. The identification of each compound was carried out by interpreting the accurate mass spectra information provided by MS and MS/MS and information previously reported in the literature. In this sense, a total of 49 compounds were detected, among which 28 were found in both extracts. This fact could be due to the differences in the extract production or even to the detection limits in the chromatographic analysis above all when the extracts are enriched in some specific compounds.Since it is important to have a better interpretation of the diversity of available phytochemicals contained in the bioactive ingredients from industrial olive byproducts, both O20 and O30 extracts were comprehensively characterized by HPLC-QTOF in order to analyze the polar bioactive fraction. The analytical platform provided two base peak chromatograms (BPC) that are shown in All compounds were classified into four major categories, oleuropein and its related compounds, iridoids, flavonoids and other compounds.40, 42, 44, 45) and its derivatives, but also the simple phenol hydroxytyrosol (13), a precursor of oleuropein, and verbascoside (32), a conjugated glucoside of hydroxytyrosol and caffeic acid [29, 34), oleuropein glucoside  and oleuropein aglycone . Other derivatives, such as methoxyoleuropein (39) and another new related compound (48), were also identified. The last one detected at m/z 763 and molecular formula C36H44O18, which gave an MS/MS spectrum with main fragment ions at m/z 539 and 307, corresponded to oleuropein and its major product ion, respectively. To the best of our knowledge, this is the first time that this derivative has been reported, although a similar compound was previously described at m/z 864 by Cardoso et al. [43) was also detected in both extracts as well as two isomers of elenolic acid glucoside . In a nutshell, tyrosol, hydroxytyrosol and oleuropein have been suggested as possible cosmetic ingredients due to their antioxidant and anti-inflammatory properties and claims of antiaging and hydration effects [It is known that the most abundant group of compounds in olive leaves is secoiridoids, mainly oleuropein (eic acid . It is weic acid . For thi effects .10); aralidioside (12), which has been reported in woody perennials for showing cardioprotective effects [21). These last two iridoids have been described for the first time in olive leaves in this work.This group comprises a wide number of monoterpenes and glucoside derivatives, whose structure may be derived from iridane. In addition, iridoids are the precursors of secoiridoids by opening the pentacyclic ring . In the  effects ; and ace17), allobetonicoside (16) and other related compound (15) with a shared fragmentation pattern at m/z 181 and 161 as main fragments (20) was also only detected in O20. However, a secologanol derivative from Gentiana depressa [m/z 687 and tentatively identified as depressine (25).Moreover, a group of iridoid glucoside derivatives was only found in O20 extracts. These compounds were tentatively identified for the first time in olive leaves as eriobioside ; paniculin (26), an isoflavone substituted by hydroxy groups that has been firstly reported in this work; two flavonols (kaempferol diglucoside (27) and rutinose (36)); and two flavones that were luteolin glucoside isomers (37 and 41).Flavonoids are among the main components of olive leaves presenting several biological properties . Six com1) and citric (3) acid, and different sugars . Moreover, simple phenols, namely, vanillin (4), which is commonly identified in olive leaves [6); and the phenolic glycoside leunoriside A (7) were also characterized. The last one, leunoriside A, was reported as a stronger antioxidant than 3-tert-butyl-4-hydroxyanisole in Leonurus japonicus [Eleocharis dulcis) [9, 11 and 22) were detected at m/z 389. These compounds are present in a high amount in olive flowers [Among this heterogeneous group, it was possible to detect organic acids, such as gluconic  . In addi flowers .24), a terpene glycoside that was described in Robusta coffee [o-glycosyl compounds were detected at m/z 401 (23) and 415 . Among these primeveroside derivatives, 28 and 30 corresponding to phenetyl primeveroside isomers, these compounds have been previously found in olive leaves and reported for their capacity to inhibit AMP-activated protein kinase (AMPK) activity, an important regulator of cellular energy homeostasis, in a hypertrophic adipocyte model [33) and the phenylpropanoid calceolarioside A (35) were also identified, the latter having been previously reported in olive leaves, although with other mass spectra data [Globularia orientalis [m/z, molecular formula and MS/MS spectrum that were obtained in O30 extract.On the other hand, cinnamoside  was used to evaluate in vitro antiradical action by TEAC assay, giving 0.592 mmol eq. Trolox/g dry extract [•+ scavenging activity to that reported in 4/g dry extract for O20 and O30 extracts, respectively. Nunes et al. reported minor ferric reducing antioxidant power for olive pomace aqueous extract (66.38–101.51 g FeSO4/kg dry extract) compared to the present study [Regarding antioxidant capacity, both extracts showed higher values than those reported in literature ,53. The  extract ,53. Lowe extract . Other gnt study . All theThe production of reactive species provides beneficial health effects in various physiological processes, including host defense against infectious agents and cell signaling ,60. On tO. europaea fruit and leaf extract enriched with 30% oleuropein (O30) was the most effective scavenger for all ROS and RNS tested. The highest quenching efficiencies were achieved for NO● (IC50 = 1.7 µg/mL) and O2●− (IC50 = 20.1–29 µg/mL). It is noteworthy that a detailed evaluation of the in vitro radical scavenging activity of industrial olive byproduct-enriched extracts is provided for the first time in this paper, comprising an opening field of research. In fact, our values are higher than those reported in commercial micronized powdered olive leaves by Goldschmidt et al. for all ROS/RNS assays [The S assays ,62.2●− is the first radical formed during oxidative processes by NADPH oxidase and is quickly transformed into more potent reactive species [2●− scavenging efficiency decreased in the following order: gallic acid > O30 extract > O20 extract > catechin. Among samples, the best O2●− scavenger was the O30 extract (IC50 = 20.1 µg/mL), while the O20 extract displayed the lowest quenching capacity based on the highest IC50 value (IC50 = 29 µg/mL). Gallic acid (IC50 = 6.0 µg/mL) exhibited the highest O2●− scavenging ability, while catechin showed the lowest quenching capacity (IC50 = 43 µg/mL). Significant differences (p < 0.05) were observed for gallic acid and catechin, while O20 and O30 extracts were not significantly different (p > 0.05). However, both extracts were statistically different (p < 0.05) from the positive controls. These results were in line with those documented for Psidium cattleianum pulp extract (IC50 = 20.6 µg/mL) [2●−  [2●− quenching power for a methanolic extract of Indigofera cassioides leaves (IC50 = 232.0 µg/mL) [Castanea sativa and Quercus robur leaf extracts exhibited higher O2●− scavenging ability associated with lower IC50 values  [O species . The O2●6 µg/mL) . Neverthctively) ,64,65. A0 µg/mL) . On the ctively) .2O2 and chloride ions catalyzed by myeloperoxidase enzymes abundantly present in mammalian granulocytic leukocytes [50 = 0.42 μg/mL) was the best quencher, followed by gallic acid (IC50 = 4.0 μg/mL), O20 (IC50 = 33 μg/mL) and O30 extracts (IC50 = 34 μg/mL). The extracts presented similar results evidenced by the absence of statistical differences (p > 0.05). No significant differences were observed for catechin and gallic acid. Otherwise, both extracts displayed significant differences (p < 0.05) towards positive controls. Similar HOCl scavenging capacities were reported for Citharexylum solanaceum pulp, skin and seed extracts (IC50 = 22.8–64.0 μg/mL), as well as Caryocar villosum pulp (IC50 = 3.6–299.0 μg/mL) and P. cattleianum skin extracts (IC50 = 32.0 μg/mL) [C. sativa leaves (IC50 = 63.8 µg/mL) [HOCl is a powerful reactive species which results from the reaction between Hukocytes . Concern0 μg/mL) ,61,68. M8 µg/mL) .● is a reactive species generated by the conversion reaction of L-arginine to L-citrulline catalyzed by nitric oxide synthase. This reactive species is involved in various physiological processes, including inflammation and immune response, and may be used as a precursor of peroxynitrite (ONOO−), which is a more damaging reactive species [50 values of extracts and positive controls in this scavenging assay increased in the following order: gallic acid < catechin < O20 extract < O30 extract, with gallic acid (IC50 = 0.20 µg/mL) and catechin (IC50 = 0.95 µg/mL) exhibiting the highest NO● quenching activity. The extracts were significantly different (p < 0.05) when compared to gallic acid and catechin. Otherwise, no significant differences (p > 0.05) were detected between both extracts that exhibited similar IC50 values . These results were in accordance with those reported for walnut leaves (IC50 = 1.95 µg/mL) [● quenching potential was observed for leaf extracts from C. sativa (IC50 = 3.10 µg/mL), Q. robur (IC50 = 3.13 µg/mL) and A. arguta (IC50 = 3.80 µg/mL) [P. cattleianum skin and pulp (IC50 = 2.2–6.8 µg/mL) and C. villosum pulp extracts (IC50 = 2.8–142 µg/mL), the obtained results were also considerably higher [● quenching power (65% of inhibition), while the other polyphenols only achieved 50% of inhibition [NO species . The IC55 µg/mL) . A lower0 µg/mL) ,67. Likey higher ,61. In ahibition .O. europaea byproduct extracts in the scavenging of ROS and RNS studied may be due to the phenolic composition, especially oleuropein as reported in the previous sections, whose scavenging capacity has already been described in previous studies [The promising outcomes of  studies ,71.O. europaea flowers showed interesting elastase and collagenase inhibition potentials [50 = 55 ± 1 μg/mL for O20 and IC50 = 100.1 ± 0.8 μg/mL for O30, which are higher than those reported previously for other plant extracts [Concerning the cosmetic potential evaluation of olive byproducts, it is noteworthy that studies in the literature are scarce, although surprisingly, the number of olive-based cosmetic products in the market is very high. Several studies demonstrate that olive byproducts possess antipigmentation potential inhibiting tyrosinase and other cosmetic properties. For example, the tentials . In facttentials ,74. By ctentials . In the extracts . In O20 extracts . Similarextracts . The proThe effects of O20 and O30 extracts on skin cell lines are of utmost importance to evaluate the potentialities of these extracts as active cosmetic ingredients. Keratinocytes are the major cells found in the epidermis, which is the outermost skin layer and where cosmetics are applied . The viap > 0.05) were detected between the extracts and medium exposure. Nevertheless, at the highest concentration tested (1000 μg/mL), the cell viability decreased to 61.05 and 42.06%, respectively, after exposure to O20 and O30 extracts. The differences observed between O20 and O30 may be due to the different bioactive compounds extracted, as reported in ®) and ex vivo skin permeation through Franz diffusion cells.The HaCaT cell line viability did not decrease after exposure to different concentrations of O20 and O30, showing a viability increase of around 100% until a concentration of 100 μg/mL. Until this concentration, no significant differences , iridoids , paniculin (26) and other compounds (7 and 24). All detected compounds were demonstrated to possess a strong potential against oxidative stress and aging as reflected in bioactivity performed assays. Specifically, O30 showed higher values in all experiments, except in HOCl scavenging and hyaluronidase inhibition, where O20 obtained the best results. Regarding cell viability assays, between 0.1 and 100 µg/mL, O20 and O30 did not lead to a decrease in viability. However, at the highest concentration tested (1000 µg/mL), both extracts resulted in viabilities of around 42 and 62%, respectively.In the present study, a robust HPLC-QTOF platform was employed to identify a total of 49 compounds; among them, 28 were found in both O20 and O30 extracts. A higher abundance of oleuropein and its related compounds was found in O30, whereas O20 was characterized by a major number of iridoids. Moreover, this work reported for the first time in olive leaves the presence of oleuropein-related compound (The promising potential of industrial olive byproducts as cosmetic ingredients demonstrated in this work is a great starting point for future perspectives that can be concluded with these industrial oleuropein-rich extracts from olive byproducts as bioactive ingredients, allowing the development of new added value products under a biocircular economy model.


Streptomyces sp. strain DBT34, which was previously demonstrated to have plant growth-promoting (PGP) and antimicrobial properties, may also have a role in protecting plants against several stresses through the production of antioxidants. The present study was designed to characterize catalase and superoxide dismutase (SOD), two enzymes involved in the detoxification of ROS, in methanolic extracts derived from six endophytic actinobacterial isolates obtained from the traditional medicinal plant Mirabilis jalapa. The results of a preliminary screen indicated that Streptomyces sp. strain DBT34 was the best overall strain and was therefore used in subsequent detailed analyses. A methanolic extract of DBT34 exhibited significant antioxidant potential in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays. The cytotoxicity of DBT34 against liver hepatocellular cells (HepG2) was also determined. Results indicated that methanolic extract of Streptomyces sp. strain DBT34 exhibited significant catalase and SOD-like activity with 158.21 U resulting in a 55.15% reduction in ROS. The IC50 values of a crude methanolic extract of strain DBT34 on DPPH radical scavenging and ABTS radical cation decolorization were 41.5 µg/mL and 47.8 µg/mL, respectively. Volatile compounds (VOC) were also detected in the methanolic extract of Streptomyces sp. strain DBT34 using GC-MS analysis to correlate their presence with bioactive potential. Treatments of rats with DBT34 extract and sitagliptin resulted in a significant (p ≤ 0.001) reduction in total cholesterol, LDL-cholesterol, and VLDL-cholesterol, relative to the vehicle control and a standard diabetic medicine. The pancreatic histoarchitecture of vehicle control rats exhibited a compact volume of isolated clusters of Langerhans cells surrounded by acinies with proper vaculation. An in-vivo study of Streptomyces sp. strain DBT34 on chickpea seedlings revealed an enhancement in its antioxidant potential as denoted by lower IC50 values for DPPH and ABTS radical scavenging activity under greenhouse conditions in relative comparison to control plants. Results of the study indicate that strain DBT34 provides a defense mechanism to its host through the production of antioxidant therapeutic agents that mitigate ROS in hosts subjected to biotic and abiotic stresses. Reactive oxygen species (ROS) and other free radicals cause oxidative damage in cells under biotic and abiotic stress. Endophytic microorganisms reside in the internal tissues of plants and contribute to the mitigation of such stresses by the production of antioxidant enzymes and compounds. We hypothesized that the endophytic actinobacterium  They are generated as a byproduct of normal metabolism, including respiration and photosynthesis -Methyl compound showed anticancer activity as observed in our isolate DBT34 and could be responsible for cytotoxic potential against HepG2 cancer cell lines. A derivative of 4-Methoxybenzeneacetic acid has been reported to be neuroprotective, as well as possessing anticancer activity  anion. Then, the mixture was kept in the dark for 15 min to allow the formazan to form. Absorbance was subsequently recorded at 560 nm using a Multiscan UV/Vis spectrophotometer and used to calculate the level of SOD activity present in the extract.The six crude extracts were dried using a rotary evaporator and diluted to 50 µg/mL in phosphate buffer (pH-8.0) and evaluated for catalase activity using a UV-Vis spectrophotometer. Hydrogen peroxide  reagent. Gallic acid was used to generate a standard curve in the range of 10–1000 mg/mL. A 200 µL volume of assay sample was prepared by combining 10 µL of sample extract with 90 µL FC reagent. This solution plus 100 µL of 15% of sodium carbonate were added to individual wells of 96-well plates. TPC was quantified as mg of Gallic acid equivalents (GAE) . The totThe antioxidant potential of a methanolic extract of strain DBT34 was determined using a free radical DPPH scavenging assay and ABTS radical cation decolorization assay.TM GO, Thermo Scientific, MA, USA). [The ability of a methanol extract of DBT34 to scavenge the DPPH free radical was determined by using the stable 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH). Briefly, for the DPPH scavenging assay, 100 µL of different concentrations (10–100 µg/mL) of the methanolic extract was placed in 96-well plate and mixed with 200 µL of DPPH solution (0.1 mM). The samples were kept in the dark for 39 min after which absorbance was recorded at 517 nm using a multi-scan UV/Vis spectrophotometer . . AscorbiA, USA). .2. All of the analyses were carried out as described by Tan et al. [Three cancer cell lines  were obtained from NCCS, Pune, India. Cancer cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) media  amended with 10% bovine serum, 100 µg/mL of streptomycin, 5 µg/mL of amphotericin B, and 100 IU/mL of penicillin and cultured at 37 °C in a humid environment and 5% atmospheric COn et al. .4 cells per 100 μL of media were placed in 96-well plates. The plates were cultured in an incubator at 37 °C and 5% CO2 for 24 h. Initial cytotoxicity of a 500 μg/mL concentration of the methanolic extract was tested against all three cell lines and the results indicated that the extract had significant cytotoxicity against HepG2 cells. Based upon the preliminary results, the extract was diluted to different concentrations , and their cytotoxicity against HepG2 cells was evaluated. Untreated cells served as a control. The methodology of the MTT assay was previously described by Tan et al. [The MTT test was used to measure the cytotoxicity of the methanolic extract of DBT34 against three cancer cell lines. A total of 1.0 × 10n et al. .Streptomyces sp. strain DBT34 was used to identify VOCs using GC-MS. The GC-MS (Clarus 680) column was packed with Elite 5MS and had a size of 30 m × 0.25 mm ID × 250 μmdf. The compounds present in the extract were separated using Helium gas as a carrier at a constant flow of 1 mL/min. The instrument temperature was 260 °C when 1 μL of extract sample was injected. The oven temperature was set as follows: 60 °C (2 min), followed by 300 °C, increasing at a rate of 10 °C min−1. Samples were exposed to 300 °C for 6 min. The mass detector conditions were as follows: 240 °C for transfer line and ion source temperature, and the ionization mode electron impact was set at 70 eV, with a scan time and interval of 0.2 s and 0.1 s, respectively. Obtained spectra were compared with a local copy of the NIST (2008) library database.A methanolic extract of The commonly prescribed DPP-4 inhibitor drug, sitagliptin, was administered as a 25mg tablet by calculating dose (2.5 mg/kg/day), along with a tablet containing 4 mg dexamethasone. Both drugs were purchased from a local pharmacy.Healthy Wistar rats, weighing 170–190 gm, were maintained in polypropylene enclosures at a standard photoperiod (14 h light: 10 h dark) under controlled environmental conditions (26 ± 1 °C) and fed a standard laboratory feed  with water ad libitum. Animals were maintained as per the guidelines of the CPCSEA , Ministry of Environment, Forest and Climate Change, Govt. of India . Approval date by CPCSEA was 29.03.2019.Type 2 diabetes was induced by providing a high sucrose diet and the oral administration of dexamethasone (corticosteroid) at a calculated dose of 1.0 mg/Kg for three weeks, as described in previous studies ,65,66. LDPP-4 inhibition by the microbial extract and by sitagliptin was assessed based on the cleavage of Gly-Pro-p-nitroanilide by the DPP-4 enzyme resulting in the production of a stable chromophore. DPP-4 inhibition activity of the microbial extract and sitagliptin was assessed by determining the release of 4-nitroaniline from the assay mixture comprising 0.1 M Tris-HCl (pH 8.0) and 2 mMGly-Pro p-nitroanilide (substrate). The assay mixture was kept at 37 °C, and the reaction was terminated by the addition of sodium acetate buffer (pH 4.5). Absorbance was recorded at 405 nm using a UV-VIS Spectrophotometer ,68. PercSerum insulin , HbA1C , glucoseHOMA indices, including HOMA-IR (insulin resistance), HOMA-β (β-cell function), and insulin sensitivity (IS) were calculated using a formulated equation in Excel developed by the Diabetes Trials Unit (DTU), a registered UK Clinical Research Collaboration Clinical (UKCRC), and a registered Clinical Trials Unit ,77.HOMAAntioxidant enzymes, including catalase, glutathione (GSH), superoxide dismutase (SOD), and lipid peroxidase (LPO) in serum, were determined using standard methods ,80,81,82Overnight fasted animals were autopsied under mild anesthesia, and the pancreas of the sacrificed rats were collected for histopathological examination. The collected pancreases were fixed in 10% formalin and subjected to gradual dehydration , clearing, and paraffin embedding. Tissues were sectioned at 5 µ; sections were placed on glass slides, deparaffinized, and stained (H&E) using a standard protocol ,86,87. CStreptomyces sp. strain DBT34 on the antioxidant activity in chickpea plants, an in-vivo experiment was conducted which involved treating chickpea seedlings with DBT-34 and letting the plants grow for four weeks. After that time, a methanolic extract (50–500 µg/mL) was prepared from the untreated four-week-old chickpea plants (control) and chickpea plants treated with Streptomyces sp. strain DBT34. The methanolic extract was used to conduct a DPPH radical scavenging assay and ABTS radical cation decolorization assay [To determine the effect of on assay ,63.p < 0.05) was used to determine statistically significant differences between the experimental materials and the controls using SPSS software.A Tukey’s multiple range test (


Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether  The first member of this family of photoreceptor proteins, bacteriorhodopsin, was discovered in halophilic archaea and functions as a light-driven outward H+ pump . Microbi+ pump , eukaryo+ pump . Upon il+ pump , ion cha+ pump . All michalophilic archaea, Halobacterium halobium; this gene influenced rhodopsin gene expression and was named the bacterio-opsin-related protein (brp) (blh) gene was also reported in Halobacterium sp. NRC-1   to clarify whether A. minutum produces retinal for the construction of a functional rhodopsin in the absence of the blh gene.We herein conducted a functional analysis of a rhodopsin and chemical analyses of pigments from the cosmopolitan freshwater Actinobacterium isolate, A. minutum KNCT was originally isolated from a river in Japan  modified from NSY medium (–1 m–2) or dark conditions at 25°C for 20 days. Cell growth was measured based on assessments of optical density at 660 nm (OD660) every 2 days using a spectrophotometer . After an incubation for 10 days, cells were centrifuged in preparation for the measurement of XLR proton-pumping activity and subsequent analyses.g at 4°C for 8 min. Cells were then washed three times with 100 mM NaCl and resuspended in 6 mL of 100 mM NaCl. The light source was a 300-W xenon lamp , and 6 mL of the cell suspension was initially placed in the dark and then irradiated for 5 min through 450-±10-nm, 520-±10-nm, and 580-±10-nm bandpass filters . The light intensity of each wavelength was fixed at approximately 7 mW cm–2 using an optical power meter  with an optical sensor . pH was measured using a pH meter . Proton-pumping activity was defined as the slope in the 30 s following the onset of illumination. This initial slope was normalized to that obtained prior to illumination. The first measurement was performed without external retinal, and the second measurement was conducted after the addition of retinal dissolved in 100% ethanol . All measurements were performed at 4°C.Light-driven proton-pumping activity was measured according to the method described by A. minutum DNA fragment encoding XLR  was chemically synthesized with codon optimization for E. coli by Eurofins Genomics. This gene fragment was inserted into the NdeI and XhoI sites of the pET21a vector (Novagene); consequently, the plasmid encoded AmXLR with hexahistidine at the C terminus. The plasmid was transformed into E. coli strain C41 (DE3) (Lucigen). E. coli cells with the plasmid were initially incubated at 37°C on an LB-medium agar plate supplemented with ampicillin , and were then incubated at 37°C in 100 mL of 2×YT medium supplemented with ampicillin  as a pre-culture. After pre-culturing, cells were incubated at 37°C in 100 mL of modified NSY medium, which was also used for the incubation of A. minutum KNCT. Protein expression was induced by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when OD660 reached approximately 0.4. All-trans retinal (Sigma-Aldrich) was added to one flask  and not to the other flask . Rhodopsin-expressing cells were collected by centrifugation , washed three times, and then re-suspended in 100 mM NaCl. A 300-W MAX-303 xenon lamp was used as the light source, and 6 mL of the cell suspension was initially placed in the dark and then irradiated through the 520-±10-nm bandpass filter for 5 min. pH was monitored using a LAQUA F-72 pH meter. The experiment was repeated using cells incubated with retinal under the same conditions after the addition of the protonophore CCCP  to inhibit proton transport. All measurements were performed at 4°C.The A. minutum cells used for the spectroscopic analysis were incubated under the same conditions as those outlined above for the measurement of proton-pumping activity. Approximately 300 mL of modified NSY medium was concentrated by centrifugation at 7,000×g at 4°C for 8 min. Half of the resulting pellet was suspended in buffer containing 50 mM Tris-HCl and 0.1 M NaCl (pH 7.0) and then ultrasonicated for 80 cycles of 1 min on and 1 min off . The supernatant was then obtained by centrifugation at 35,600×g at 4°C for 10 min, followed by ultra-centrifugation at 178,000×g at 4°C for 30 min  to separate the pellet containing the membrane fraction. The pellet was homogenized on ice and suspended in Tris-HCl buffer at pH 7.0. Hydroxylamine was added to the suspension to a final concentration of 100 mM, and absorption spectra were measured every 10 min using the ultraviolet-visible spectrophotometer UV-2450 with the ISR2200 integrating sphere (Shimadzu). The reaction constant of the reaction, τ, was estimated from a single exponential fit:The y=y0+A*exp(–x/τ)where y and x are the absorption change and reaction time, respectively.A. minutum cells used in the HPLC-MS/MS analysis were incubated under the same conditions as those described above. Cells in approximately 300 mL of culture medium were pelleted by centrifugation at 7,000×g at 4°C for 8 min, and the supernatant was completely removed. In the HPLC-MS/MS analysis, aliquots of acetone were added to the frozen pellet in a microtube, which was then placed in an ice-cooled ultrasonication bath for pigment extraction. Samples were then ultrasonically homogenized for a few minutes, and acetone supernatants were immediately separated from the particulate material by centrifugation and directly injected into the HPLC apparatus for analysis.–1. All mobile phases were degassed in vacuo using ultrasonication. Mobile-phase reservoir bottles were designed to prevent any contact between the mobile phases and air during the analysis. The solvents used for the HPLC mobile phases included LC/MS-grade distilled water and methanol purchased from Kanto Chemical and LC/MS-grade formic acid and HPLC-grade acetone purchased from Waco Pure Chemical Industries. APCI was set to the following conditions: nebulizer gas flow, 2.2 L min–1; interface temperature, 320°C; desolvation line temperature, 140°C; heat-block temperature, 250°C; drying gas flow, 3 L min–1. The parameters of the Q1 scan in the positive ion mode of the mass spectrometer were set as follows: scan range, m/z 100.00–400.00; event time, 0.200. Parameters for the detection of retinal during multiple reaction monitoring (MRM) in the positive ion mode were as follows: precursor ion ([M+H]+), m/z 285.25; product ion, m/z 160.10; dwell time, 10 ms; collision energy (CE), –10 V.The HPLC-MS/MS instrument consisted of a Shimadzu Nexera X2 liquid chromatography system, comprising a CBM-20A communication bus module, two DGU-20A3R/5R HPLC degassing units, three LC-30AD solvent delivery units constituting a ternary pumping system, an SIL-30AC autosampler, CTO-20AC column oven, and LCMS-8030 triple quadrupole mass spectrometer connected through an atmospheric pressure chemical ionization (APCI) interface (Shimadzu). The system was then coupled to a personal computer configured to run Shimadzu LabSolution software. Reverse-phase HPLC was performed under the following conditions: column, Zorbax Eclipse Plus C18 ; eluent, the ternary gradient program summarized in A. minutum cells were incubated for 20 days under light or dark conditions. No significant differences were observed in the growth of A. minutum between the light and dark conditions until the mid-log phase  or dark (cell-DARK). A light-induced pH change was observed in both cell-LIGHT and cell-DARK samples in the absence of exogenous retinal  E. coli does not produce retinal, and (3) modified NSY medium did not contain retinal. Therefore, A. minutum constructs a functional rhodopsin, the XLR apoprotein of which binds to an endogenous chromophore.Light-induced pH changes in suspensions of  retinal A despiteA. minutum. In the analysis of XLR proton-pumping activity in cells incubated for 10 days, the initial slope of cell-LIGHT with an external supply of retinal was significantly higher than that of cell-DARK with an external supply of retinal  only work with all-trans retinal. Therefore, we assumed that AmXLR has an all-trans retinal as a chromophore. However, A. minutum produced sufficient retinal under different culture conditions. H. salinarum. This increase was greater in a brp gene deletant (that only possessed the blh gene), indicating that not all rhodopsin are functional, even though this archaeon produces retinal. In other words, the amount of retinal may be less than that of rhodopsin apoproteins among some rhodopsin-possessing prokaryotes.Microbial rhodopsins have several retinal isomers, such as all-A. minutum KNCT produced retinal, the genes responsible for its biosynthesis have not yet been identified. Among rhodopsin-possessing Actinobacteria, a lycopene cyclase encoded by the crtYc/Yd genes catalyzes the conversion of lycopene to β-carotene, which is then supplied for endogenous retinal  and Micrococcus luteus (accession numbers: AYO50480 and AYO50481), to which the putative genes of A. minutum are closely related; however, they are less closely related to the crtYc/Yd clusters . Therefore, R. lacicola may also produce retinal through an unknown pathway similar to that in A. minutum. However, the present results do not exclude the possibility of a retinal biosynthesis pathway without the C50 carotenoid as an intermediate. While a β-carotene 15,15′ dioxygenase cleaves one β-carotene to two retinal, asymmetrical cleavage may convert one C50 carotenoid to one retinal, such as 15,15′-monooxygenase, encoded by the diox1 gene. In that case, the number of retinal molecules produced by asymmetrical cleavage is half the number of retinal molecules produced by dioxygenase cleavage. This may be one of the reasons why A. minutum only functionalizes some of the XLR expressed.Although we showed that clusters . It has  retinal . FurtherA. minutum KNCT, which possesses a rhodopsin gene and lacks the known retinal biosynthetic genes blh and diox1, are functional. The cleavage of β-carotene is not an abiotic reaction, but an enzymatic reaction that produces retinal  retinal using an unknown retinal biosynthetic gene(s) for functionalizing rhodopsin. Although rhodopsins in prokaryotes that lack blh or diox1 were previously considered to be non-functional, the endogenous production of retinal was confirmed in this strain. Interestingly, diverse organisms that include strains belonging to the phyla Deinococcus-Thermus, Firmicutes, Proteobacteria, Bacteroidetes, Chloroflexi, and Euryarchaeota have putative rhodopsin genes, but lack blh and diox1 (data not shown); however, it currently remains unclear whether these organisms endogenously produce retinal without the currently known retinal biosynthetic genes. Further studies on these strains may reveal how widespread retinal production is in blh-lacking strains and also how these rhodopsin-possessing prokaryotes obtain retinal to construct functional rhodopsins.In conclusion, we herein report that some XLR rhodopsins in the freshwater Actinobacterium,  retinal ; therefoMicrobes Environ 35: ME20085.Nakajima, Y., Kojima, K., Kashiyama, Y., Doi, S., Nakai, R., Sudo, Y., et al. (2020) Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins. https://doi.org/10.1264/jsme2.ME20085Supplementary Material


Metabolic syndrome, obesity and type 2 diabetes are metabolic disorders characterized by the insulin resistance and the impairment in the insulin secretion. Since impairment in the oxidative stress and adipocyte metabolism contribute to the formation of obesity and diabetes, targeting adipose tissue can be considered as an effective approach to fight against them. Rosiglitazone is used for treatment for patients with type 2 diabetes via inducing lipogenesis and transdifferentiation of white adipose tissue into brown adipose tissue. Since the development of such therapeutics is required to control the formation and function of brown fat cells, we aimed to reveal possible molecular mechanisms behind rosiglitazone induced biochemical changes in the adipose tissue.Cells were expanded in the adipocyte culture medium supplemented with 5 µg/mL insulin following 2 days' induction. After those cells were treated with rosiglitazone 0, 0.13 mol/L and 10 µmol/L rosiglitazone for 48 hours and at 8th day, cells were collected and stored at -80 °C. Then the cells were used to evaluate antioxidant enzyme activities, mineral and trace element levels and fatty acid composition.Glucose-6-phosphate dehydrogenase and glutathione reductase significantly reduced in rosiglitazone-treated groups compared to the control. Na, Mg, K, Ca, Cr, Fe, Ni, Cu, Zn, Rb, Sr, Cs, Ba and Pb were determined in the cell lysates via ICP-MS. Also, relative FAME content decreased in the rosiglitazone-treated groups compared to the control.Rosiglitazone treatment at low doses showed promising results which may promote brown adipose tissue formation. Adipose tissue stores and/or releases fatty acids (FAs) depends on the need of the energy requirement of organisms since adipocytes are the main regulators of energy and glucose homeostasis Rosiglitazone (RSG) is one of the drugs used for patients with type 2 diabetes to reduce insulin resistance and hyperglycemia via decreasing blood glucose levels. Also, this drug induces adipose differentiation, triglyceride storage (TG) and lipogenesis T2DM, metabolic syndrome and obesity are characterized by the insulin resistance and the impairment in the insulin secretion which both are tightly associated with the impairment in the adipose tissue functions, and increased levels of reactive oxygen species (ROS) termed as oxidative stress +), oxidized glutathione (GSSG), glutathione reductase (GR), reduced glutathione (GSH), sodium azide, hydrogen peroxide (H2O2) and ethylenediaminetetraacetic acid (EDTA), nicotinamide adenine dinucleotide phosphate (NADP+), magnesium chloride (MgCl2), Tris [Tris (hydroxymethyl) aminomethane] were purchased from Sigma-Aldrich (USA). Chemicals and organic solvents used for lipid analysis were obtained as HPLC grade. Hexane, 65% HNO3 of SUPRAPUR® grade, methanol and HCl were purchased from Merck (Germany). C4-C24 FAME mix standard solution and methyl tricosanoate were obtained from Sigma-Aldrich (USA) and used for FAME quantification, identification and as internal standard, respectively.Protease inhibitor cocktail was obtained from Sigma-Aldrich (Germany). Glucose-6-phosphate (G6P), sodium phosphate monobasic and dibasic, 6-phosphogluconate (6-PG), reduced nicotinamide adenine dinucleotide phosphate  fractions. SV cells were grown, and adipocyte differentiation was performed as described by Serkan et al. Cells were scraped off in 1 mL PBS and then transferred into 1 mL centrifuge tubes. Samples were centrifuged at 2000 rpm for 5 minutes, and then cell pellets were suspended in the 200 mmol/L sodium phosphate buffer (pH 7.4) containing protease inhibitor cocktail and sonicated for 10 seconds on the ice. Afterwards, samples were centrifuged at 14,500 rpm for 25 minutes at 4 °C and stored at -80 °C freezer until experiments.2, 0.6 mmol/L G6P and 0.2 mmol/L NADP+ in 100 mmol/L Tris/HCl buffer (pH 8.0). G6PD activity was evaluated spectrophotometrically by following NADPH production at 340 nm and 37 °C +/min under the indicated assay conditions was used to define one unit (U) of activity. Specific G6PD enzyme activity was represented as the number of units/mg protein.The assay mixture was prepared with 10 mmol/L MgCl6-Phosphogluconate dehydrogenase activity (G6PD) was determined by the same method as described above for the G6PD. Only, 0.6 mmol/L 6G6P was substituted for 0.6 mmol/L 6-PG as a substrate in the assay mixture (23).6-Phosphogluconate dehydrogenase activity (G6PD) was determined by the same method as described above for the G6PD. Only, 0.6 mmol/L 6G6P was substituted for 0.6 mmol/L 6-PG as a substrate in the assay mixture Modified Staal method was used to evaluate GR activity at 37 °C spectrophotometrically. The incubation mixture was prepared with 200 mmol/L sodium phosphate buffer (pH 7.4), 1 mmol/L GSSG, 0.2 mmol/L NADPH and cell lysate as enzyme source. The decrease in the absorbance at 340 nm was monitored for 60 seconds Glutathione-S-transferase enzyme activity was determined by measuring the conjugation of GSH with 1-chloro-2, 4-dinitrobenzene (CDNB) as described by Habig et al. μL tissue lysate and completed to 500 μL with double distilled water 2O2. Afterwards, GPx activity was measured spectrophotometrically for 30 seconds at the room temperature.Incubation mixture contained 200 mmol/L potassium phosphate buffer (pH 7.4), GR enzyme (10 U/mL), 400 mmol/L sodium azide, 200 mmol/L EDTA, 100 mmol/L GSH, 2 mmol/L NADPH, 5 BCA method was used to determine protein concentrations of the samples by using Spectramax M2 microplate reader. The assay was performed with the Pierce™ BCA Protein Assay Kit by following instructions Cell lysates were diluted by a factor of 10 in 65% HNO 3 of SUPRAPUR® grade. Trace and mineral element levels were determined by using Agilent 7700x ICP-MS  as described by Aydemir et al. In situ acidic direct transesterification (DT) was used for derivatization of fatty acids according to Gomez et al. Fatty acid methyl esters were analyzed by 7890-B Agilent GC equipped with a 5977A Agilent MSD detector and a DB-23 column (60 m × 0.25 mm × 0.25 mm film thickness). 1 mL FAME extract was used from each sample. The splitless mode was applied, and the injector temperature was set up to 250 °C. Helium was used as the carrier gas at 50 °C and 180 kPa (33 cm/S). The column temperature was increased from 50 °C to 175°C at 25 °C per min and then from 175 °C to 235 °C at 4 °C per min, and the detector temperature was 250 °C.All data were analyzed by GraphPad Prism, and one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test for multiple comparisons for comparison data sets. All data were represented as the mean ± standard deviation (SD).G6PD, GR and GPx activities decreased in the RSG treated cells compared to the control, however, this decrease was not significant for GPx activity. On the other hand, there was not a substantial change in the GST and 6-PGD activities , Table 1Na, Mg, K, Ca, Cr, Fe, Ni, Cu, Zn, Rb, Sr, Cs, Ba and Pb were determined in the cell lysates via ICP-MS. Ca and Mg levels significantly decreased in the 0.1 µmol/L RSG treated groups compared to the 1 µmol/L treated ones. Also, K concentrations increased in the RSG-treated groups compared to the control, where Na levels significantly decreased in control and 0.1 µmol/L RSG treated groups compared to the 1 µmol/L treatment groups .On the other hand, Ba and Rb levels significantly decreased in 0.1 µmol/L dosage groups compared to the other groups, where Fe levels elevated considerably. Pb, Cs and Zn concentrations decreased in the RSG treated groups in comparison with the control. Sr and Ni levels had their peaks in 0.1 µmol/L RSG treated groups, where Cs had peak concentration in 1 µmol/L RSG dosage groups .Relative FAME content decreased in RSG-treated groups compared to the control a and relMetabolic disorders, T2DM and obesity are characterized by the impairment in the insulin secretion, sensitivity and adipose tissue function. Moreover, both oxidative stress and lipid metabolism play a significant role in the pathogenesis of these diseases and targeting fat tissue can be considered as a good strategy to treat these diseases. Rosiglitazone is used to treat T2DM via reducing insulin resistance and hyperglycemia, also inducing the conversion of WAT into BAT G6PD, GR and GPx activities decreased in RSGtreated groups compared to the control , Table 1+2 levels significantly increased in the 0.1 µmol/L RSG treatment compared to the 1 µmol/L group (ATP) channels play a vital role in the insulin secretion via depolarization of the cell membrane that leads to the Ca+2 influx in the cell, causing insulin secretion. Inhibition of ATP-sensitive potassium (KATP) channels by RSG was reported before Afterwards, we evaluated mineral and trace element levels in control and RSG-treated samples. Our data showed that Ca/L group . ElevateConcentrations of various trace elements including cadmium, chromium, iron, nickel, silver, caesium, lead, rubidium and zinc were investigated in patients with T2DM. Increased Cr and Zn levels were reported in T2DM patients in vivo studies since they increase the thermogenic capacity of WAT Furthermore, we investigated total FAME and fatty acid composition in RSG treated and control groups. Our data have revealed that 0.1 µmol/L RSG treatment reduced relative FAME content, where 1 µmol/L treatment did not show any change in the FAME content a. We shoAccording to our data, tricosanoic acid, 11eicosenoic acid, 11,14-eicosadienoic acid, docosanoic acid, 10-heptadecenoic, 10-pentadecenoic acid and γ-linolenic acid significantly decreased upon RSG treatment compared to the control . Since tOverall, RSG treatment reduced G6PD and GR enzymes contributing to the oxidative stress and fatty acid metabolism. Also, RSG treatment improves trace element and mineral levels that enable restoring of insulin resistance and BAT formation. Relative FAME content reduced upon RSG treatment and relative content of some fatty acids which promote BAT formation increased. Thus the influence of fatty acid composition and types on the BAT formation should be further investigated.Adipose tissue is considered as a target for the treatment of insulin resistance and diabetes. RSG is used for the treatment of T2DM and induces transd-ifferentiation of WAT into BAT via increasing mitochondrial mass and lipid oxidation. Therefore, the development of therapeutics targeting adipocytes requires information about the molecular mechanisms controlling the formation and function of brown fat cells. RSG treatment on the adipocytes at low doses showed promising results which may address conversion of WAT into BAT. However, the impact of fatty acids on the brown fat formation should be further investigated.Acknowledgement. We thank Dr Serkan KIR for primary mouse adipocytes. The authors gratefully acknowledge the use of the services and facilities of the Koc University Research Center for Translational Medicine (KUTTAM) and Koc University Tupras Energy Center (KUTEM).The authors state that they have no conflicts of interest regarding the publication of this article.


Nesfatin-1 was identified as a satiety factor involved in the regulation of metabolism. Altered levels of circulating nesfatin-1 had been observed in a variety of diseases characterized by energy imbalance. However, there was no published data about nesfatin-1 levels in acromegaly.We evaluated serum nesfatin-1 levels in 13 patients with acromegaly at baseline and postoperatively, and in 21 age- and body mass index (BMI)-matched healthy subjects.P = .004). There was a positive correlation of serum nesfatin-1 levels with diastolic blood pressure  and homeostasis model assessment of insulin resistance (HOMA-IR)  in patients with acromegaly. While a successful surgery decreased serum GH levels, the serum nesfatin-1 levels did not change in acromegaly (P = .965). At last, we compared serum GH/nesfatin-1 levels with predictive markers for aggressive behaviors in pituitary adenomas. There was no relationship between serum nesfatin-1 levels and tumor's size, Ki-67 index, mutant p53, or MGMT proteins. However, increased serum GH levels were positively correlated with tumors’ size (P = .023) and mutant p53 proteins expression (P = .028).Compared with the healthy subjects, patients with acromegaly had significantly increased levels of serum insulin, high-density lipoprotein cholesterol, triglyceride, and growth hormone (GH). Moreover, the acromegaly group had nesfatin-1 levels higher than controls (1.96 ± 0.56 ng/mL vs 0.61 ± 0.10 ng/mL, Circulating nesfatin-1 was increased in acromegaly, which was involved in metabolism regulation. The mean body mass index (BMI) was 24.33 ± 0.39 kg/mGroup 2  13 patients with acromegaly  were involved in the study. The diagnosis of acromegaly was based on the clinical presentation, magnetic resonance imaging (MRI) image and the serum levels of GH. While 10 patients were newly diagnosed, 3 patients experienced tumor recurrence after at least one operation. Additionally, 2 patients showed the invasion of cavernous sinus by the tumor with MRI. These 5 patients’ tumors were classified as aggressive and invasive pituitary adenomas.All the blood specimens for hormone or biochemical analysis were drawn from a forearm vein in the morning after overnight fasting. Postoperational blood samples were collected on the third day postoperatively. Tumor samples used in the study were obtained fresh from operation at Sanbo Brain Hospital. Informed consent was obtained from all patients prior to the study. All experiments using human tissues are approved by the Institutional Review Board of Sanbo Brain Hospital.2.2g for 10 minutes at 4 °C, and stored at −80 °C until assay. Serum nesfatin-1 levels were investigated using Enzyme Linked Immunosorbant Assay . The linear range of the assay was 0.78 to 50 μg/L. The detection limit was increased by adding 0.78 ng/mL pure nesfatin-1 to the samples and subtracting this from the total amount measured at the end of the experiment, as we previously did.Blood samples were collected after an overnight fast, separated immediately by centrifugation at 1000 × 2.36-methylguanine DNA methyltransferase (MGMT) (1:150 Invitrogen) were then applied overnight at 4 °C. The cutoff values were 3% for nuclcar- associated antigen Ki- 67, 5% for p53, and 10% for MGMT respectively.Immunostaining was performed on 4-μm-thick sections of formalin-fixed and paraffin-embedded samples. The sections were dewaxed by xylene, dehydrated by gradient ethanol, and then rehydrated with PBS . To facilitate antigen retrieval, the sections were incubated in 10 mM of citric acid (pH 6.0) at 120 °C for 150 seconds. We used 3% hydrogen peroxide to neutralize endogenous peroxidases of the samples for 15 minutes. Primary antibodies against p53 (1:100 Invitrogen), nuclcar- associated antigen Ki-67 (1:200 Invitrogen), and O2.42. The homeostasis model assessment of insulin resistance (HOMA-IR) was studied using following equations (HOMA-IR = fasting insulin (mU/L) × fasting glucose [mmol/L]/22.5). The quantitative insulin sensitivity check index (QUICKI) was calculated as followed (QUICKI = 1/[log fasting glucose {mg/dL} + log fasting insulin {mU/L}]).The tumor size was evaluated by the MRI before the operation. BMI was calculated as weight (kg)/height (m)2.5U test. Differences between hormonal levels before and after operation were made with the paired t test. The correlations between different parameters were performed with the simple regression analysis. The predictive value of serum nesfatin-1 for acromegaly was analyzed by receiver operating characteristic (ROC) curve analysis. P< .05 was taken as statistically significant.Data were expressed as mean ± SEM. All data analysis was carried out by Statistical Product and Service Solutions 22.0. Comparisons between 2 groups were analyzed using the Mann–Whitney 33.1P = .089) There was no significant difference in systolic pressures and diastolic pressures among the 2 groups. Acromegalic patients had a higher fasting plasma glucose levels compared with the normal subjects (Table P = .000) and nesfatin-1 level  in the acromegaly group  or HOMA-IR  in the acromegaly group  However, postoperative serum nesfatin-1 levels showed a margin significant correlation with serum GH levels .Serum GH levels in acromegaly decreased significantly after operation. (38.67 ng/mL vs 12.82 ng/mL, 3.3We further related the serum nesfatin-1 and GH levels to the characteristics of the growth hormone-secreting pituitary adenoma.4In this study, our patients with acromegaly suffered from disordered metabolism, and had an elevated levels of nesfatin-1 meanwhile. The increased levels of nesfatin-1 in acromegaly might due to 2 reasons: the tumor itself may secret nesfatin-1; alternatively the disordered metabolism caused the increased levels of this hormone. Moreover, the surgical treatment did not normalize the circulating levels of nesfaitn-1 3rd days postoperatively. The half-life of nesfatin-1 was 9 to 10 minutes.[Consequently, the elevated levels of circulating nesfatin-1 were a compensatory mechanism to normalize the disordered metabolism in acromegaly. In addition to the regulation of metabolism, nesfatin-1 also participated in cardiovascular actions and gastric mobility, stress, and reproduction.[Compared with the control group, our acromegalic patients showed a significant positive correlation of nesfatin-1 with diastolic pressure. The increased plasma nesfatin-1 levels were observed in hypertension patients, and associated with a higher risk of hypertension.Recent studies suggested a role of nesfatin-1 in the development of tumor. The intratumoral nesfatin-1 was heavily expressed in colon cancer,Still our study placed importance of curing endocrine diseases in acromegaly. The disordered metabolism in acromegaly might promote tumor progression by causing some hormones imbalance.Our study has, however, several limitations. First, the limited cases in our study might be a shortcoming. Second, we haven’t explored the exact mechanism. Sample size may cause some results to be insignificant or irrelevant and more studies should detect the role of nesfatin-1 in acromegaly.In conclusion, we first recorded the increased levels of circulating nesfatin-1 in acromegaly. The altered levels of nesfatin-1 were most likely to be caused by the energy imbalance. At last, we suggested that GH excess could promote the progression of pituitary adenomas.Conception and design were contributed by CY and NL. Collection and assembly of data were contributed by YY, SH, ZY and PW. Data analysis and interpretation were contributed by YY, SH, ZY and PW. Manuscript writing was contributed by all authors. All authors gave final approval of the manuscript.


Zika virus (ZIKV) infection during pregnancy was associated with microcephaly in neonates, but clinical and experimental evidence indicate that ZIKV also causes neurological complications in adults. However, the changes in neuron-glial communication, which is essential for brain homeostasis, are still unknown. Here, we report that hippocampal slices from adult rats exposed acutely to ZIKV showed significant cellular alterations regarding to redox homeostasis, inflammatory process, neurotrophic functions and molecular signalling pathways associated with neurons and glial cells. Our findings support the hypothesis that ZIKV is highly neurotropic and its infection readily induces an inflammatory response, characterized by an increased expression and/or release of pro-inflammatory cytokines. We also observed changes in neural parameters, such as adenosine receptor A2a expression, as well as in the release of brain-derived neurotrophic factor and neuron-specific enolase, indicating plasticity synaptic impairment/neuronal damage. In addition, ZIKV induced a glial commitment, with alterations in specific and functional parameters such as aquaporin 4 expression, S100B secretion and glutathione synthesis. ZIKV also induced p21 senescence-associated gene expression, indicating that ZIKV may induce early senescence. Taken together, our results indicate that ZIKV-induced neuroinflammation, involving nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NFκB) pathways, affects important aspects of neuron-glia communication. Therefore, although ZIKV infection is transient, long-term consequences might be associated with neurological and/or neurodegenerative diseases. ZIKV markedly affects the cytoarchitecture of the brain, as well as induces neurochemical and molecular dysfunctions in neural cells6. Current clinical and experimental data indicate that ZIKV also affects the mature brain, causing neurological complications including Guillain-Barré and other myelitis and encephalitis-like illnesses11. However, the underlying neurochemical changes are still poorly understood. The hippocampus is a crucial brain structure for learning and memory, and hippocampal damage leads to the progression of severe psychological and cognitive impairments. Hippocampal neurogenesis, which persists throughout life, is affected by immune activity triggered by pathogens including ZIKV12.Zika virus (ZIKV) is a single-stranded RNA flavivirus, originally identified in Uganda in 1947, which has currently received attention because may cause severe damage in the fetal brain following maternal infection during gestation. In Brazil, the 2015 ZIKV outbreak was associated with microcephaly in neonates exposed to the virus during pregnancy13. Astrocytes are able to sense and respond to changes in their surrounding microenvironment15. These cells also contribute to the formation and integrity of blood–brain barrier (BBB), affect axonal outgrowth and support neuronal metabolism and synaptic activity17. Moreover, astrocytes actively participate in neuron-glial communication, releasing trophic factors and inflammatory mediators19. With regard to ZIKV infection, astrocytes are potentially the first cells targeted by ZIKV in the CNS, since they present the AXL receptor, a candidate receptor to host ZIKV in the brain21. Indeed, microglial cells are the tissue-resident macrophage-like immune cells that continuously maintain an immune surveillance of the CNS22, thus they can mediate inflammatory response after ZIKV exposure. The production of pro-inflammatory cytokines and other signalling molecules by microglia and/or astrocytes is part of the defence system against invading pathogens in the brain and are particularly orchestrated by nuclear factor κB (NFκB) pathway24. Despite of both astrocytes and microglia presenting protective/defensive roles, these cells can shift their phenotype and release a wide range of harmful signalling molecules. As glial cells regulate tissue recovery after injury, dysfunctions in these cells may be associated with neurological outcomes of ZIKV and represent a risk factor for neurodegenerative diseases and neuropsychiatric disorders26.Although neurons are the main focus of neuroscience research, glial cells are essential for the central nervous system (CNS), particularly because alterations in their functions can promote neuronal survival or deathIn line with this, the aim of this study was to evaluate the changes in neuronal and glial functions in an ex vivo experimental model of acute hippocampal slices from adult rats submitted to ZIKV infection. Thus, we determined neurochemical parameters, inflammatory response, cellular and molecular pathways, including nuclear factor erythroid 2-related factor 2 (Nfr2) and NFκB, which are strongly associated with neuron-glia communication. This interaction is a crucial process for CNS homeostasis, and our findings provide the first evidence that ZIKV alters neuron-glia communication.2 to 106 plaque-forming units (PFU) as shown in the Fig. 6 PFU  and interleukin (IL) 1β release  in the hippocampal slices  2 and 4, which can bind virus, bacteria, pro-inflammatory cytokines and alarmins, such as HMGB112. However, only TLR2 expression was increased by ZIKV  2, which increased with ZIKV exposure Table . As such29. Conversely, mRNA expression levels of the enzymes superoxide dismutase (SOD) 1 and 2 decreased, while inducible nitric oxide synthase (iNOS) increased. In addition, the expression of Nrf2 decreased in a time-dependent manner, as well as the mRNA levels of heme oxygenase 1 (HO1), a fundamental defence mechanism for cells exposed to challenge stressors  Table . This in30. However, sirtuin 1 (SIRT1), a pathway that may counteract the inflammatory response and oxidative redox imbalance, did not change.Besides these pathways, Table Neurotrophic factors have been shown to modulate synaptic plasticity, neural response and recovery. ZIKV induced a fast increase in brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) release, as well as vascular endothelial growth factor (VEGF) mRNA levels Table . However31. In our experimental model, we observed that ZIKV increased only the expression of adenosine receptor A2a  and other specific markers such as glutamate transporters (GLAST and GLT1) and glutamine synthetase (GS) did not change in the acute presence of ZIKV  Table , whose rRegarding neurons, although there was an increase in extracellular NSE levels, there was no alteration in the expression of other specific markers, such as the EAAC1 glutamate transporter, N-methyl-D-aspartate receptor 1 (NMDA-R1), synaptophysin and β-tubulin III, after ZIKV exposure Table .10. Herein, for the first time, we reported that ZIKV is able to quickly infect hippocampal slices from adult rats, acutely causing a wide range of cellular and molecular alterations, regarding to redox, inflammatory and neurotrophic parameters  expression and in the GSH content, probably as an early compensatory mechanism in response to ZIKV exposure. In contrast, ZIKV acutely modulated the expression of other genes related to redox homeostasis/oxidative stress; particularly, there was a downregulation of HO1, SOD1 and SOD2, and an upregulation of iNOS. Accordingly, a recent study in human iPSC-derived astrocytes showed that ZIKV infection induced oxidative stress, mitochondrial failure and DNA damage2. ZIKV-induced dysfunctions in mitochondrial activity are also potentially associated with excitotoxicity. Notably, ZIKV-infected neurons release increased levels of glutamate37. Although we did not observe changes in the expressions of astrocytic and neuronal glutamate transporters, their activity may be impaired by oxidation45, potentially causing excitotoxicity. In addition, this process is potentiated by Ca2+ release from the mitochondria and endoplasmic reticulum, and ZIKV can interfere with Ca2+ uptake by mitochondria2. However, at least acutely, ZIKV did not modulate NMDA-R1 protein levels.Astrocytes play an important role in the CNS antioxidant defence, since they can provide glutathione (GSH) and superoxide dismutase (SOD) to neurons12. In the context of acute hippocampal injury, neurotrophic factors have been shown to modulate neural response and recovery46. The decrease of BDNF could contribute to impair synaptic plasticity. However, synaptophysin, a pre-synaptic protein widely used as a marker of synaptic plasticity, was not affected at this short time. In addition, considering that A2a receptor is associated with synaptic plasticity and inflammatory process48, the increased expression of this receptor induced by ZIKV, can be a possible link between ZIKV, neuroinflammation and long-term neurological diseases26. It is important to note that the decrease in BDNF and increase in A2a perhaps favour the release of glutamate in neurons49, a common event associated with excitotoxicity and age-related diseases. In this sense, ZIKV-induced an upregulation of the senescence marker p21 in hippocampal slices. Moreover, the protein S100B is frequently used as a marker of astrocyte activation, and can produce either neurotrophic or deleterious effects, depending on the concentration50, showed a decreased release, suggesting that ZIKV also affects trophic signalling mediated by astrocytes.The differential expression profile observed for TLRs may indicate differences in infection and immunity in response to ZIKV. Although TLRs trigger inflammatory and antiviral responses, they can also modulate adult hippocampal neurogenesis51. Increasing evidence has suggested that activation of Nrf2 is more restricted to astrocytes52. While the increased expression and activity of Nrf2 are associated with protective mechanisms, deficiencies have been correlated with exacerbated astrogliosis, GFAP expression and worsening of inflammatory parameters in a mouse model of neurodegeneration, as well as with impaired neuronal differentiation of neural stem cells in the subgranular zone of the hippocampus53. Furthermore, in the CNS, cell-type specific pathological roles of NFκB have been described, including aberrant synapse to nuclear communication in neurons, and glial activation leading to chronic neuroinflammation, with consequent neuronal cell death28. Considering that there is an interplay between inflammatory and oxidative signals, in which Nrf2 depletion enhances NFκB signaling, and the latter eventually modulates Nrf2 transcription51, data indicate that the transcription factors, NFκB and Nrf2, may be important mechanistic partners in the altered neuron-glial communication observed after ZIKV exposure.Since there is a close relationship between inflammation and redox signalling, we investigated classical pathways that interconnect these events in neural cells, namely NFκB and Nfr2. Nfr2 is a transcription factor involved in the adaptive response to cellular stress, including the oxidative stress induced during the inflammatory response, and their target genes, which induce antioxidant enzyme production, GSH synthesis and eventually inhibit cytokine-mediated inflammation54. Moreover, and just as importantly, our study has generated data to indicate that ZIKV-induced neural damage occurs in the mature brain, particularly in the hippocampus. Based on GFAP expression and content, astrocytes may appear to be unaffected by acute ZIKV exposure. However, other specific and functional parameters such as AQP4, S100B secretion, GSH biosynthesis, and underlying glial signalling pathways indicate acute glial commitment  and YFV vaccinal strain 17DD (produced by Instituto de Tecnologia em Imunobiológicos Bio-Manguinhos), were propagated in African green monkey kidney E6 (VERO E6) cells (ATCC CRL-1586). Viruses were also titrated on VERO E6, using a standard plaque assay. Viral stocks were maintained at − 80 °C.Brazilian ZIKV strain 17  were obtained from the breeding colony of the Department of Biochemistry  and maintained under a controlled environment . All animal experiments were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committees of Federal University of Rio Grande do Sul (process numbers 24419 and 36949) and Instituto de Cardiologia/Fundação Universitária de Cardiologia (CEUA-IC/FUC 001/2018).56. Slices were then transferred immediately into 24-well culture plates (one slice per well), with each well containing 0.3 mL of physiological saline medium . The medium used for slices was exchanged every 15 min with fresh saline at room temperature (equilibration period) for up to 60 min. It is important to note that this is an acute model of hippocampal slices, which are viable up to 5 h after preparation57.The hippocampi were dissected from brain and transversally sliced (0.3 mm thickness) using a Mcllwain Tissue Chopper2–106 or an equivalent volume of non-infection control medium) or YFV  for an adsorption period of 1 h at 37 °C. Subsequently, the inoculum was exchanged with fresh saline medium and hippocampal slices were maintained at 37 °C for 1 h or 2 h. The extracellular medium and slices were harvested at these two time points for subsequent analyses.Following the equilibration period, the medium of hippocampal slices was removed and replaced with 0.3 mL fresh saline containing different PFU of ZIKV  of slices using a commercial UV assay from Bioclin (Brazil). Results are expressed as percentages of the non-infection control value.Neuron-specific enolase (NSE) activity extracellular NSE was measured using an eletrochemiluminescent assay purchased from Roche Diagnostics. The assay is a double sandwich that uses an antibody anti-NSE bound with ruthenium, which produces light emission when excited. The reaction and quantification were performed by the equipment Elecsys-2010 (Roche Diagnostics Corporation). Results are expressed as percentages of the non-infection control value.31. The results are expressed in pg/mL and the average minimum sensitivity of the ELISA kits detection was: 25.0 pg/mL for TNFα; 12 pg/mL for IL1β; 16 pg/mL for IL6; 3 pg/mL for IL10; and 5 pg/mL for MCP1.Cytokine levels were measured in the extracellular medium using ELISA kits for TNFα (Peprotech), IL1β, IL6, IL10 and MCP1 (Invitrogen)31. The results are expressed in pg/mL. The ELISA kits detect a minimum of 12 pg/mL for BDNF and 31.2 pg/mL for GDNF.BDNF and GDNF levels were measured in the extracellular medium, using commercial ELISA kits from Invitrogen and R&D Systems, respectively58. Briefly, 50 µL of extracellular medium from slices and 50 µL of Tris buffer were incubated for 2 h on a microtiter plate previously coated with monoclonal anti-S100B . Next, the samples were incubated with polyclonal anti-S100B (Dako) for 30 min, and then, peroxidase-conjugated anti-rabbit antibody (Amersham) was added for a further 30 min incubation period. A colorimetric reaction with o-phenylenediamine (Sigma-Aldrich) was observed at 492 nm. Results are expressed as percentages of the non-infection control value.S100B secretion was measured by an enzyme-linked immunosorbent assay, as previously described58. Hippocampal slices were suspended in a 100 mM sodium phosphate buffer with 140 mM KCl (pH 8.0) containing 5 mM EDTA. After that, protein was precipitated with 1.7% metaphosphoric acid. The supernatant was assayed with o-phthaldialdehyde  at room temperature for 15 min. Fluorescence was measured using excitation and emission wavelengths of 350 and 420 nm, respectively. A calibration curve was performed with standard GSH (Sigma-Aldrich) solutions at concentrations ranging from 0 to 500 μM. Results are expressed as percentages of the non-infection control value.GSH levels were fluorometrically assessed as previously described59; RP-YFV and FP-YFV60). Amplification was carried out in a StepOne Real-Time PCR system  under the following conditions; uracil DNA glycosylase (UDG) incubation at 50 °C for 2 min; initial denaturation and Platinum Taq activation at 95 °C for 2 min, followed by 40 cycles of amplification (15 s at 95 °C and 30 s at 60 °C). All real-time assays were performed in triplicate and results depict the means of these triplicate values. Viral RNA quantification was estimated in relation to ZIKV or YFV standard curves (108 to 10 copies) and data analysis was performed with the StepOne software v2.2.2.Total RNA was isolated from hippocampal slices (control and infected) using TRIzol Reagent . Extracted RNA (1 μg) was submitted to cDNA synthesis by High Capacity cDNA Reverse Transcription Kit . Quantitative PCR for viral copies determination was performed in total volumes of 12.5 µL containing 3 µL of cDNA, 6.25 µL of 2× Platinum quantitative PCR SuperMix-UDG (Invitrogen—Life Technologies), 200 nM each of forward and reverse primers .For neural gene expression, the messenger RNAs (mRNAs) encoding TNFα (#Rn99999017_m1), TNF receptor 1 , IL1β (#Rn00580432_m1), IL1 receptor type I , HMGB1 (#Rn02377062_g1), COX2 (#Rn01483828_m1), TLR2 (#Rn02133647_s1), TLR4 (#Rn00569848_m1), NFκB p65 (#Rn01502266_m1), NFκB p50 (#Rn01399572_m1), Nrf2 (#Rn00582415_m1), HO1 (#Rn01536933_m1), iNOS (#Rn00561646_m1), SOD1 (#Rn00566938_m1), SOD2 (#Rn00690588_g1), GCL (#Rn00689046_m1), PI3K (#Rn01769524_m1), Akt (#Rn00442194_m1), p21 (#Rn 00589996_m1), SIRT1 (#Rn01428096_m1), VEGF (#Rn01511602_m1), adenosine receptors A1 (#Rn00567668_m1), A2a (#Rn00583935_m1), A2b (#Rn00567697_m1), A3 (#Rn00563680_m1), GFAP (#Rn00566603_m1), vimentin (#Rn00667825_m1), nestin (#Rn00564394_m1), AQP4 (#Rn00563196_m1), GLT1 (#Rn00691548_m1), GLAST (#Rn00570130_m1), GS (#Rn01483107_m1), EAAC1 (#Rn00564705_m1), and β-actin (#Rn00667869_m1) were quantified using the TaqMan real-time RT-PCR system using inventory primers and probes purchased from Applied Biosystems (Thermo Fisher Scientific), as referred for each gene3VO4, 10 mM NaF and 50 mM Tris–HCl (pH 6.8). Samples were separated by SDS/PAGE (20 μg protein per sample) and transferred to nitrocellulose membranes using a semi dry blotting apparatus . The membranes were blocked with 2% albumin in Tris-buffered saline with Tween 20 (T-TBS) and then incubated overnight (4 °C) with anti-PI3K , anti-Akt , anti-NMDAR1 , anti-GFAP , anti-synaptophysin , anti-tubulin βIII , or anti-actin . β-actin was used as a loading control. Subsequently, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG  for 1 h. The chemiluminescence signal was detected in an Image Quant LAS4010 system  using an ECL kit . Results are expressed as percentages relative to non-infection control conditions.Hippocampal slices were lysed in a solution containing 4% SDS, 2 mM EDTA, 1 mM NaP < 0.05 were considered significant. Heatmaps were created using GraphPad Prism 7 and Heatmapper61.Differences among groups were statistically analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s test. All analyses were performed using the GraphPad Prism 7 . Values of Supplementary Information.


Attention deficit hyperactivity disorder (ADHD) is a childhood-onset chronic neurobehavioral disorder, with multiple genetic and environmental risk factors. Chronic inflammation may be critical for the progression of ADHD. An Shen Ding Zhi Ling (ASDZL) decoction, a traditional Chinese medicine prescription, is clinically used in ADHD treatment. In this study, we investigated the effects and underlying anti-inflammatory mechanisms of ASDZL in young spontaneously hypertensive rats (SHRs), a widely used model of ADHD. SHRs were divided into the SHR model group (vehicle), atomoxetine group (4.56 mg/kg/day) and ASDZL group (21.25 g/kg/day), and orally administered for four weeks. Wistar Kyoto rats were used as controls (vehicle). We found that ASDZL significantly controlled hyperactivity and impulsivity, and improved spatial memory of SHRs in the open field test and Morris water maze test. ASDZL reduced the pro-inflammatory factors interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 and increased anti-inflammatory factor IL-10 in SHRs, and decreased the activation of microglia, astrocytes and mast cells in the prefrontal cortex (PFC) and hippocampus. Furthermore, the results indicated that ASDZL inhibited the neuroinflammatory response by protecting the integrity of the blood-brain barrier and suppressing the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB signaling pathways of SHRs. In conclusion, these findings revealed that ASDZL attenuated ADHD symptoms in SHRs by reducing neuroinflammation. Attention deficit hyperactivity disorder (ADHD) is a chronic neurobehavioral disorder with a worldwide prevalence of 1.4–3.0%. The core symptoms of ADHD are inattention, motor hyperactivity and impulsivity, which affect the full lifecycle . SimilarResearches have proposed an association between ADHD and inflammatory mechanisms. Several studies have shown that patients displaying ADHD symptoms have abnormal serum cytokine levels compared with normal controls . In addiEfficacious medications such as methylphenidate and atomoxetine (ATX) are widely used, often in combination with complementary psychosocial approaches. However, their effectiveness has been questioned because they do not meet the broader and, especially, long-term clinical needs of many ADHD patients. Therefore, scientific and clinical studies are challenging current conceptions of the etiology and pathogenesis of ADHD that might alter future clinical approaches .An Shen Ding Zhi Ling (ASDZL) is an empirical formula prescribed by Professor Xinmin Han for treating ADHD, which has been used for more than 20 years in Jiangsu Provincial Hospital of Traditional Chinese Medicine and included in Chinese Medical Clinical Pathway for Treating ADHD . ASDZL cHowever, the neuroinflammatory mechanisms of ADHD and the effect of ASDZL on the condition remain unclear. Prefrontal cortex (PFC) and hippocampus dysregulation has been found in ADHD patients and animal models . SpontanThe ASDZL decoction was composed of 12 herbs, as shown in The herbs in ASDZL were mixed and soaked in 8-time volume of distilled water (v/w) for 1 h. Then the herbs were boiled twice in 8-time volume of distilled water (v/w) for 1 h each time. The supernatants acquired from both decoctions were pooled and concentrated to 0.71 g/ml of crude herbs using rotavapor .A Waters Aquity H-Clas ultra-high performance liquid chromatograph was used and chromatographic separation was achieved by the AQUITY UPLC HSS T3 chromatographic column . The column oven temperature was set at 25°C and the mobile phases consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B). The elution conditions were as follows: 0 min, 10% B; 3 min, 10% B; 26 min, 90% B; 30 min, 10% B, at the flow rate of 0.2 ml/min. The linear trap quadrupole (LTQ) ORBITRAP XL tandem mass spectrometer (MS) was applied and MS conditions were: ion source, heated electrospray ionization (HESI); scan ranges, 100–1,000 m/z; collision energy, 35. The positive ionization mode used the following parameters: heater temperature, 300°C; sheath gas flow rate, 45 arb; aux gas flow rate, 10 arb; spray voltage, 4 kV; capillary temperature, 300°C, capillary voltage, 35 V; tube lens, 110 V. The negative ionization mode used the following parameters: heater temperature, 300°C; sheath gas flow rate, 45 arb; aux gas flow rate, 10 arb; spray voltage, 4 kV; capillary temperature, 300°C; capillary voltage, −35 V; tube lens, −110.00 V. The reference standards used were Baicalin, Saikosaponin A, Rhynchophylline, Isorhynchophylline, Emodin , Curcumin, Quercetin, Phyllyrin, Catalpol, Acteoside , and the detailed chemical information of ASDZL was shown in n = 10) and SHRs (n = 30) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  2016-0006) and maintained on a 12-h light/dark cycle under the temperature of 23 ± 2°C and relative humidity of 50–60% in the experimental animal center of Nanjing University of Chinese Medicine. All rats received adaptive feeding for 1 week prior to administration. All procedures were carried out in accordance with the National Institute of Health Guidelines for Laboratory Animals, and were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (No. 201910A041). The weight changes of rats in each group were shown in Three-week-old male WKY rats (n = 10 per group): SHR group (model group), ATX group (4.56 mg/kg), ASDZL (21.25 g/kg) group, and WKY rats were used as the control group. There were no significant differences in the weight gain of rats among the groups  reuptake inhibitor and commonly used drug for ADHD treatment  was usede groups , and no e groups . The WKYThe OFT was conducted at 0, 2 and 4 weeks of treatment, and the Morris Water Maze (MWM) test was performed after 3 weeks of treatment. Behavioral tests were performed based on previously established methods  with minm), time spent in the central area (s) and rearing times were recorded using Easy-Tracking System . The OFT was conducted in a quiet environment from 8:00–12:00 am. The scent marks were removed between tests.The OFT is used to assess behavioral performance in ADHD models . The opeThe MWM test is widely used for assessing the learning and memory abilities. The MWM apparatus consisted of a circular black iron tank (150 cm in diameter and 50 cm in depth) filled with water (24 ± 2°C), a video camera above the arena and a computerized tracking system. A circular black platform with a rough surface (12 cm in diameter) was submerged approximately 1 cm below the water surface. Spatial acquisition trials were conducted during day 1–5 with four trials per day, and the trials were conducted from four positions. Each rat had 60 s to find the platform. Rats that failed to locate the platform were guided there and allowed to rest on it for 10 s. The probe trials were conducted on day 6 without the platform. The escape latency (s) in spatial acquisition trials, annulus visits and time spent in the target (southeast) quadrant (s) in the probe trials were recorded using the Easy-Tracking System.The concentrations of interleukin (IL)-1β, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in the serum, PFC and hippocampus were measured using ELISA kits purchased from MULTI SCIENCES, China or Shanghai WESTANG, China, according to the manufacturer's protocol. In addition, about 6–8 ml blood can be collected from abdominal aorta of each rat and 40 or 100 μL serum was needed for each target protein tested by ELISA.3) of each rat was submerged into electron microscope fixing solution at 4°C for 3 h, washed three times with PBS (pH 7.4), fixed with 4% osmium acid, and washed with PBS again. Then the samples were dehydrated in gradient alcohol washes, embedded in Epon 812 and polymerized at 60°C for 48 h. The samples were sliced into ultrathin sections of 60–80 nm, which were negatively stained with uranyl acetate and lead citrate and examined using a JEM-1200EX electron microscope to observe the ultrastructure of the BBB in different groups.The PFC . The ImageJ 1.52v software was used to analyze number of positive stained cells in the sections.NF-κB (IκBα) , IκBα , p-NF-κB , NF-κB , and GAPDH . Following incubation with secondary antibodies. The ChemiDoc™ MP Imaging system  was used to quantify the protein bands with the Image Lab software . Each experiment was repeated at least three times.The proteins were extracted from the PFC or hippocampus tissue in each group and measured by the BCA protein assay kit . Equal amounts of proteins were separated with SDS-PAGE, and transferred to polyvinylidene difluoride membranes . The membranes were blotted overnight at 4°C with primary antibodies: anti-ZO-1 , occludin , p-p38 , p38 , p-c-Jun N-terminal kinase (JNK) , JNK , p-inhibitor of post hoc test to determine the statistical significance, which was performed by GraphPad Prism 6.0 . Differences with p < 0.05 were considered statistically significant.Data were presented as mean ± standard deviation (SD) and were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s The main components of ASDZL were determined by UPLC and compared with the reference standard solution. A total of 10 chemical components in ASDZL were determined, namely Catalpol, Acteoside, Isorhynchophylline, Baicalin, Rhynchophylline, Phyllyrin, Quercetin, Saikosaponin A, Curcumin and Emodin .ASDZL had no negative effect on the weight changes and development of rats, as shown in The data revealed that there was a decrease in escape latencies of all four groups during the spatial acquisition trials. The ATX and ASDZL groups spent less time to locate the escape platform compared with SHR group on the day 4 and 5 . The SHRp > 0.05, IL-1β, IL-4, IL-6, TNF-α and MCP-1 are pro-inflammatory factors commonly used to evaluate inflammatory responses, and IL-10 is an anti-inflammatory factor . As showAs shown in As shown in p-JNK, p-IκBα and p-p65 NF-κB levels were significantly increased in the PFC and hippocampus of SHRs, while their total levels did not obviously change. And the p-p38 MAPK, p-JNK, p-IκBα and p-p65 NF-κB levels in ATX and ASDZL groups were significantly decreased compared with those in SHR group after 4 weeks of gavage treatment  disorders and has been thought to be related to ADHD. Previous studies found abnormal expression of inflammatory factors in ADHD patients . HoweverHealthy cerebral blood vessels have a functional BBB which protects the surrounding brain parenchyma from the invasion of immune cells and deleterious stimuli in the blood . The ECsMAPKs are important for intracellular signal transduction and play vital roles in regulating neuroplasticity and inflammatory responses, and p38 MAPK and JNK are important members of the MAPK family, which have been considered as anti-inflammation targets . Inhibit-3β/β-catenin pathways to play a role in DA signaling in postsynaptic membrane .YS and XH contributed to conception and design of study. HY, TC, and ML contributed to acquisition of data. TC and SL contributed to analysis of data. YS and HY contributed to drafting the manuscript. SL and ML contributed to revising the manuscript critically for important intellectual content. All authors approved the version of the manuscript to be published.This work was financially supported by National Natural Science Foundation of China , and Postgraduate Research and Practice Innovation Program of Jiangsu Province, China (Grant No. KYCX20_1466). Project of Natural Science Research of the Higher Education Institutions of Jiangsu Province, China (No. 20KJB360001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.