Lingonberry leaves and fruits are associated with a range of potential bioactivities related to their phenolic content and composition, but the identification of major biological activity markers remains limited. The present study aimed at the isolation of lingonberry phenolic fractions and biological activity evaluation of them. Crude dry extracts of lingonberry leaves and fruits were fractionated by chromatography using Sephadex LH-20 and analyzed by validated HPLC-PDA method. For each fraction, the anticancer activity against human clear cell renal cell carcinoma (CaKi-1), human colon adenocarcinoma (HT-29), and human malignant melanoma (IGR39) cell lines was determined using MTT assay, and the radical scavenging, reducing, and chelating activities were investigated using ABTS, FRAP, and FIC assays, respectively. Further, 28 phenolics were identified and quantified in the crude extract of lingonberry leaves and 37 in the extract of fruits. These compounds, during fractionation steps, were selectively eluted into active fractions, enriched with different groups of phenolics—monophenols, anthocyanins, phenolic acids, catechins, flavonols, or proanthocyanidins. Fractions of lingonberry leaves and fruits, obtained by the last fractionation step, proved to be the most active against tested cancer cell lines and possessed the greatest antioxidant activity. In this perspective, the predominant compounds of these fractions—polymeric and mainly A-type dimeric proanthocyanidins—also quercetin can be considered to be anticancer and antioxidant activity markers of lingonberries. Vaccinium vitis-idaea L. (lingonberry), spontaneous growing evergreen shrubs (family Ericaceae Juss.), are well known for their health-protective properties and potential use in the treatment of urinary tract infections and chronic diseases, associated with free radicals damage in the human body. Fruits and leaves of this plant are considered to be a good source of phenolic compounds, which ensure various mechanisms of the antioxidant defense system [e system ,2. Majore system ,4. Sciene system . Phenolie system ,7,8. Phee system ,9,10,11.A number of studies have been conducted on lingonberries’ raw materials phytochemical characterization and pharmacological evaluation, but there are challenging problems that arise in this domain ,12,13,14One way to overcome these problems is to purify or fractionate the crude extracts. The development of a fractionation methodology may improve the elucidation of the phytochemical and pharmacological characterization of lingonberry phenolics. Evaluation of the biological properties of fractions with prevailing groups of different phenolics is desirable for the selection of plants with increased levels of compounds that are pharmacologically relevant or for the development of functional food ingredients with health-protecting properties ,21,22. TAn effective gel-filtration chromatography technique using Sephadex LH-20 was applied in the present material, resulting in three characteristic fractions from the crude extract of lingonberry leaves and three from fruits, and quantified using a novel developed and validated HPLC-PDA method. To the best of our knowledge, this is the first comprehensive report on the phytochemical composition of lingonberry leaves and fruits, revealing the presence of procyanidins B1, B2, B3, A1, A2, and C1. Antioxidant activity covering three distinct mechanisms of action, namely, free radical scavenging, reducing, and chelating activities in vitro were evaluated in order to provide a full picture of antioxidant activity capacity. Three human cell lines of different origins, namely, renal cell carcinoma (CaKi-1), human colon adenocarcinoma (HT-29), and human malignant melanoma (IGR39) were selected to test proliferation inhibitory activity of obtained lingonberry fractions and standard substances of predominant lingonberry phenolics. As far as we know, no study to date has compared the anticancer effect on these different cancer cell lines and free radical scavenging, ferric reducing, and chelating activities of lingonberry leaves and fruits after fractionation of phenolic compounds.The aim of the present study was to determine the qualitative and quantitative profiles of obtained lingonberry fractions and to characterize their antioxidant activities and anticancer effect. Comprehensively characterized novel fractions with potential chemo-active phytochemicals could be further functionalized in the phytopreparations, functional food, or integrated into the phytochemotherapeutic strategies.O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), avicularin (quercetin-3-O-arabinofuranoside), guaiaverin (quercetin-3-O-arabinopyranoside), rutin (quercetin-3-O-rutinose), reynoutrin (quercetin-3-O-xyloside), kaempferol, nicotiflorin (kaempferol-3-O-rutinoside), afzelin (kaempferol-3-O-rhamnoside), astragalin (kaempferol-3-O-glucoside), trans-cinnamic acid, caffeic acid, chlorogenic acid (3-O-caffeoylquinic acid), neochlorogenic acid (5-O-caffeoylquinic acid), cryptochlorogenic acid (4-O-caffeoylquinic acid), p-coumaric acid, ferulic acid, sinapic acid, benzoic acid, vanillic acid, protocatechuic acid, arbutin, and resveratrol—were purchased from Sigma-Aldrich , whereas cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, procyanidin B3, hyperoside , and 6”-O-acetylisoquercitrin (quercetin-3-O-(6”-acetylglucoside)) were purchased from Extrasynthese .HPLC grade standard substances—procyanidins B1, B2, C1, A1, A2, (+)-catechin, (−)-epicatechin, quercetin, quercitrin (quercetin-3-3), sodium acetate, 3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), 3--2,5-diphenyltetrazolium bromide (MTT)—were obtained from Sigma-Aldrich , whereas potassium persulfate (K2S2O8), anhydrous ferrous chloride (FeCl2) were from Alfa Aesar . All standard substances and chemicals were of analytical grade.Chemicals for antioxidant or anticancer activities assays—6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ethylenediaminetetraacetic acid (EDTA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri-(2-pyridyl)-S-triazine (TPTZ), ferric chloride , penicillin/streptomycin solution (100×), and phosphate-buffered saline (PBS) were purchased from Gibco .The plasticware for cell cultures was obtained from Techno Plastic Products , Corning , and Thermo Fisher Scientific . Dulbecco’s modified Eagle high glucose medium (DMEM Glutamax), TrypLEThe following solvents were used: 99.9% acetonitrile, 99.9% acetone, 99.9% methanol, 99.9% dimethyl sulfoxide (DMSO), 99.8% anhydrous acetic acid, 37% hydrochloric acid (HCl), all of which were obtained from Sigma-Aldrich ; 99.8% trifluoracetic acid—from Merck ; and 96% ethanol—from Vilniaus degtine SC . The water used in this research was purified on a Millipore Milli-Q apparatus.2 in a humidified atmosphere. Cell cultures were grown to 70% confluence, trypsinized with 0.125% TrypLE™ Express solution before passage, and used until passage 20.Human malignant melanoma cell line (IGR39), human colon adenocarcinoma cell line (HT-29), and human clear cell renal cell carcinoma line (CaKi-1) were obtained from American Type Culture Collection . Cancer cell lines were cultured in DMEM Glutamax medium supplemented with 10% FBS and 1% antibiotics at 37 °C with 5% COPlant raw materials for research were randomly collected at the end of the fruits ripening period in September 2019 in North-East Lithuania  from different natural sites of lingonberries ) and then pooled. Fruits of lingonberries were immediately lyophilized in a ZIRBUS sublimator 3 × 4 × 5/20  in 0.01 mbar, −85 °C conditions, whereas leaves were dried at room temperature in a dark place. Freeze-dried fruits and air-dried leaves were ground with a Retsch 200 mill  to homogenous powder.p < 0.05) yields of phenolics, determined by the HPLC-PDA method, were as follows. A precise weight of 40 g of powdered lingonberry leaves was extracted with 250 mL of 80% aqueous acetone, and the same precise weight of powdered lingonberry fruits was extracted with 200 mL of 70% aqueous acetone by ultra-sonication in Elmasonic P ultrasonic bath  for 15 min. The extraction procedures were repeated four times for leaves, and three times for fruits, until complete exhaustion. Supernatants of each raw material were combined after centrifugation  in a Biofuge Stratos centrifuge . Obtained extracts were evaporated in an IKA RV 10 rotary evaporator  at 40 °C until the acetone was removed and subjected to lyophilization to get dry extracts of lingonberry fruits and leaves.Experimentally selected extraction conditions, leading to the highest  packed with already equilibrated gel filtration resin, Sephadex LH-20 , which was used as a stationary phase. Various elution ways were tested by using different amounts of water, different concentrations of ethanol, methanol, and acetone aqueous solutions as a mobile phase on the basis of previous researches ,25,26. TPhenolics in crude extracts and each fraction of lingonberries were identified and quantified by HPLC-PDA  according to Raudone et al. with some modifications . ChromatO-(4”-(3-hydroxy-3-methylglutaryl)-rhamnoside (quercetin-HMG-rhamnoside), and 2-O-caffeoylarbutin, because of commercially unavailable standards. They were tentatively quantified using calibration curves of standard substances with similar chemical structures. Limits of detection (LOD) and of quantification (LOQ) were determined via the signal-to-noise ratio method. The trueness of the method was expressed as percent recoveries of phenolics at low, medium, and high concentrations of range, each analyzed in triplicate. To assess the repeatability and intermediate precision of the method, relative standard deviation percentages (% RSD) of peak areas of each quantified phenolic compound were calculated within (six times per day) and between days (three consecutive days), respectively, resulting in total repeatability of 18 replicates.The described modified method was validated following international guidelines . The selAntioxidant activities of the crude extracts and fractions of lingonberries were evaluated by three different in vitro spectrophotometrical assays to determine the most active lingonberry phenolics and compare their predominant mechanisms on the antioxidant defense system. For antioxidant activity estimation, all testing samples were dissolved in 70% ethanol until complete dissolution, obtaining a concentration of 0.2 mg/mL.2S2O8 in darkness at room temperature for 16 h. The obtained stock solution was diluted with distilled water for the absorbance of 0.80 ± 0.02 at 734 nm, determined by spectrophotometer . The working ABTS solutions (3 mL) were mixed with test solutions—crude extracts or fractions of lingonberries (20 µL)—and the absorbance of these mixtures was measured at 734 nm after 1 h of incubation. The results were expressed as micromolar of standard antioxidant—Trolox equivalents per gram of dry weight of fractions (µM TE/g DW).ABTS radical scavenging activity was evaluated by the method described by Re et al. . Briefly3 in a final ratio of 10:1:1 (v/v/v), were mixed with test solutions (20 µL), and the absorbance was recorded at 593 nm after 1 h of incubation at room temperature [In the case of reducing activity, working FRAP solutions (3 mL), consisting of 300 mM acetate buffer, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl2 (50 µL), followed by adding 5 mM ferrozine solution (0.2 mL), incubation for 10 min at room temperature, and absorbance recording at 562 nm. Blank and negative control solutions were prepared in the same manner, only ferrozine and testing solutions, respectively, were substituted by the solvent [To assess chelating activity, testing solutions (1 mL) were mixed with 2 mM FeCl solvent . Micromo3 melanoma and renal cancer cells and 8 × 103 colorectal carcinoma cells per well) and incubated at 37 °C. After 24 h of incubation, serial double dilutions of lingonberry fractions, including crude extracts, and standard substances of phenolic compounds were made in microplates (standard substances were dissolved in DMSO). Cells treated only with the medium (or medium with 0.5% of DMSO in the case of standard substances) were used as a negative control, whereas medium without cells served as a positive control. After 72 h of incubating at 37 °C, the medium in all the wells was replaced with the fresh one containing 0.5 mg/mL of MTT. The liquid was aspirated from the wells and discarded after 3–4 h. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at 570 nm (test wavelength) and at 630 nm (reference wavelength) by multi-detection microplate reader Multiskan GO . Hill fit to lingonberry fraction or standard substance dose–cell metabolic activity (absorbance) curves were applied and results of cell viability assay were expressed as the effective concentration (EC50) values, which reduce cell viability by 50%.The effect of lingonberry fractions and standard substances of phenolic compounds on cancer cell viability was studied using an MTT assay described by Grigalius and Petrikaite . Brieflyp < 0.05). Principal components analysis (PCA) was implemented to highlight differences and classify obtained fractions of lingonberry. Correlations between variables were analyzed using Pearson’s correlation test. Data were processed using IBM SPSS Statistics version 26.0 package and Microsoft Office Excel 2016 software.All data were given as mean ± standard deviation (SD) from at least three replicates for each sample. In order to determine significant differences between values, analysis of variance (ANOVA) followed by a Tukey post-hoc test was performed. The significance of the difference was defined at the 5% level (r2 > 0.999) of the response versus concentration of each phenolic compound were achieved at minimum ranges of 1.6–50.0 µg/mL (for the majority of compounds) and a maximum range of 31.3–500.0 (for arbutin). LOD and LOQ of all tested phenolics varied in the range of 0.03–0.90 μg/mL and 0.08–2.67 μg/mL, respectively. Obtained intra-day and inter-day precision values (expressed as % RSD) were lower than the 2% threshold, with total repeatability of different phenolics varying from 0.1 to 1.7% -epicatechin. Large amounts and diversity of flavonols were also observed. Compounds of this group contributed up to 11.9% of the total identified phenolics in the crude extract of lingonberry leaves, with avicularin, hyperoside, and quercetin-HMG-rhamnoside being predominant. From the results, it is clear that flavonols of lingonberry leaves consisted mainly of the glycosides of quercetin (97.3%), whereas glycosides of kaempferol and aglycones of flavonols were not prevalent in L1. In the profile of flavonol glycosides, rhamnosides and arabinosides were predominated . Phenolic acids—prevailing chlorogenic and cryptochlorogenic acids and lower (p < 0.05) amounts of neochlorogenic and p-coumaric acids—contributed only 2.7% of the identified phenolics content and can be considered as minor phenolics of lingonberry leaves.The results of the quantitative analysis provided in p < 0.05) amounts of hydroxycinnamic acids. These compounds were effectively isolated with this fraction at the first fractionation step. As the main groups of phenolic compounds of fraction L3, flavonols, and catechins, as well as their derivatives—B-type dimeric proanthocyanidins (distributed in a ratio of 3.3:4.7:2)—can be regarded. Out of these groups, the highest (p < 0.05) amount of (+)-catechin was determined. The purest fraction of lingonberry leaves, marked L4, was achieved during the last fractionation step and distinguished by high contents of polymeric and mainly A-type dimeric proanthocyanidins. Procyanidin A1 contributed 52.8% to the total identified phenolics of this fraction and thus can be considered as the predominant phytochemical marker.The fraction of lingonberry leaves marked L2 was distinguished by a rather simple profile of phenolic compounds, basically consisting of arbutin  and much lower  amounts of procyanidins A1, B2, and quercitrin out of these groups. Flavonols in fraction F1 included quercetin derivatives (83.1%), relatively high amounts of quercetin aglycone (16.1%), and much lower (p < 0.05) contents of kaempferol derivatives. Profile of flavonols glycosides consisted of rhamnosides (52.3%), galactosides (14.7%), arabinosides (11.8%), and more than twice lower presence of other sugar moieties. Benzoic acid, followed by high levels of (+)-catechin, which made up 15.4% and 13.8%, respectively, of the total identified phenolics content, can be considered as phytochemical markers of F1. Other groups of phenolics—anthocyanins, hydroxycinnamic acids, and monophenols—were not prevalent in the obtained crude extract of lingonberry fruits.The distribution of phenolic compounds in the crude dry extract and fractions of lingonberry fruits is shown in O-galactoside, benzoic, and chlorogenic acids were regarded to be predominant phytochemical markers of this fraction. Major phenolics of fraction F3 were (+)-catechin, also quercitrin, procyanidin B1, and hyperoside. Generally, this fraction was characterized by high levels of flavonols, catechins, and B-type dimeric proanthocyanidins . It may be noted that fraction F3 was enriched with a small amount of stilbenoid resveratrol . The last fraction of lingonberry fruits, marked F4, was distinguished by the highest (p < 0.05) content of procyanidin A1, followed by procyanidin A2, and accounted for 34.5% and 25.1%, respectively, of total identified phenolics. Besides proanthocyanidins, high levels of quercetin and lower levels (p < 0.05) of kaempferol were also observed; the sum of them made up 8.7% of the identified phenolics content in this fraction.The fraction of lingonberry fruits marked F2 did not contain flavonols, catechins, proanthocyanidins, and was comprised of anthocyanins (40.8%), phenolic acids, including their precursors (56.5%), and a small amount of arbutin. Cyanidin-3-Concerning the total amounts of detected phenolic compounds within different fractions of lingonberry fruits, the following ascending order was determined: F1 < F4 < F2 < F3. Contents of phenolics in the crude extract of lingonberry fruits—F1—were even 24 times lower, compared with the most abundant fraction of lingonberry fruits—F3.O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, also chlorogenic, cryptochlorogenic, protocatechuic, p-coumaric, and other phenolic acids. The last PC3 component constituted the lowest part of the total variance (8.7%) and highly correlated with positive loadings of procyanidins C1, A1, A2, and quercetin.To classify fractions of lingonberry leaves and fruits, PCA was performed using relative amounts of the phenolic compounds as percentages of the total content of each fraction. The three principal components explaining 97.4% of the total data variance were obtained. The PC1 accounted for 56.2% of the total variance and positively correlated with procyanidins B1, B2, B3, (+)-catechin, (−)-epicatechin, quercitrin, hyperoside, avicularin, quercetin-HMG-rhamnoside, rutin, reynoutrin, and other flavonol glycosides. The second PC2 component described 32.5% of the total variance and differentiated samples containing the highest levels of arbutin, cyanidin-3-The score plots of lingonberry fractions showed their arrangement into the four groups according to closeness with principal components . The firThe results of PCA revealed that the phenolic profile of investigated fractions significantly differed from each other in the same raw material, and different fractions were distinguished by high levels of specific phenolic compounds. Meanwhile, a similar pattern of phenolics was determined between fractions of lingonberry leaves and fruits obtained from the same fractionation steps, indicating different phenolics elution with solvents of a certain polarity.p < 0.05) values determined in the crude extract of lingonberry fruits marked F1 and fraction of lingonberry leaves marked L4, respectively. A similar antiradical activity was observed between L2 and F2 fractions, and between L3, F3, and F4 fractions. In FRAP assay, L3 and F3 fractions surpassed all other fractions by the greatest (p < 0.05) reducing activity, followed by L4 and F4 fractions. Whereas the lowest (p < 0.05) reducing activity was found in the crude extract of lingonberry fruits marked F1, with almost eight and a half lower activity than average. FIC assay showed a similar trend to the results of ABTS and FRAP assays. The highest (p < 0.05) chelating activity was expressed in the fractions of L4 and F4, and the lowest (p < 0.05) one, approximately three times lower, in F1. There were no significant differences between the activity of L1, L2, and F3 fractions, while the activity of F2 was slightly higher (p < 0.05) than L2.Antioxidant activities of fractions of lingonberry leaves and fruits, including crude extracts, were variable . The radAntioxidant activity of crude extracts of lingonberry leaves and fruits or obtained fractions according to average values of all antioxidant assays were in the following ascending order: F1 < L2 ~ F2 < L1 < F3 ~ F4 < L3 ~ L4. All assays confirmed the lowest antioxidant activity of crude extract of lingonberry fruits, and the highest activity of fractions, enriched with catechins, flavonols, or proanthocyanidins, depending on the applied method and the predominant mechanism of action. It was found that the average antioxidant activity of fractions of lingonberry leaves, including crude extracts, was nearly one and a half times greater than the average antioxidant activity of fractions of lingonberry fruits.p < 0.05) anticancer activity against all three cell lines (EC50 varied from 1.1 to 1.5 mg/mL against different cell lines). Fractions L4 and V4, which were predominated with proanthocyanidins, were the most active ones . A majority of tested lingonberry fractions were more active against renal cancer cells and showed lower activity against colon cancer cell line.Lingonberry fractions of leaves and fruits, including crude extracts, reduced the viability of malignant melanoma (IGR39), colon adenocarcinoma (HT-29), and clear cell renal cell carcinoma (CaKi-1) lines . In geneFractions of lingonberry leaves were distinguished by about two times greater average values of anticancer activity compared to fractions of fruits. Anticancer activity of lingonberry fractions based on average values of anticancer activity against different cell lines was in the following anticancer activity ascending order: F1 < L2 ~ F2 < L1 ~ L3 ~ F3 < F4 ~ L4.The results provided in 50) indicate lower activity, strong negative correlations  between antioxidant and anticancer activities of lingonberry fractions were found. The strongest correlation  was found between FIC antioxidant activity and MTT assay against malignant melanoma cancer cell line.Since higher values of antioxidant activity  indicate higher activity, and higher values of cell viability assay . Values of ferric reducing activity of different fractions were also well correlated  with contents of all flavonol glycosides, catechins, and procyanidins B1, B2, B3. On the other hand, greater ferrous chelating activity was related to higher contents of procyanidins C1, A1, A2 . A similar tendency of results was obtained when assessing correlations between values from MTT assays with phenolics in different fractions. It was found that higher content of quercetin in lingonberry fractions determined higher anticancer activity and lower EC50 values against different cancer cell lines. The strongest correlation  was found between values of MTT assay against clear cell renal cell carcinoma line and quercetin contents in fractions of lingonberry leaves, followed by the strong correlation  between values of MTT assay against colon adenocarcinoma cell line and quercetin contents in fractions of lingonberry fruits. Furthermore, strong correlations between values of MTT assay against all cancer cell lines and proanthocyanidins contents in fractions of lingonberry leaves and fruits were also observed, with the strongest correlation  between anticancer activity against melanoma cancer cell line and proanthocyanidins A1, A2, C1 contents in fractions of lingonberry leaves.Strong correlations between results of assays of antioxidant and anticancer activity and specific compounds in different fractions of lingonberries were also observed. Obtained antioxidant activities were strongly correlated with contents of flavonol aglycones in lingonberry fractions, with the strongest correlation between values of ABTS assay and quercetin contents in fractions of lingonberry leaves and fruits , which were partly free from major phenolic acids, anthocyanins, arbutin, and were distinguished by an abundance of catechins and various flavonols . In compand Wang . Resveraand Wang ,41.One of the most relevant groups of compounds are proanthocyanidins. The abundance of them in herbal raw materials is highly associated with health-protecting properties . The lasVaccinium genus and are expressed in greater amounts than single chain-linked B-type. Interlinkages of subunits increase the electron delocalization capacity of the phenyl radical and are important structural features for the activity [Research on grape seed extract elucidated the numerous biological effects of proanthocyanidins and their potent antioxidant activity. Studies confirm their significance in cancer chemoprevention and inhibition . Lingonbactivity ,49. Proaactivity .Our obtained fractions L4 and F4 were more active against HT-29 cell line compared to the grape seed extract, which is also rich in proanthocyanidins, as revealed by Kaur et al. Grape seed extract at 50 µg/mL concentration reduced the number of live HT-29 cells by only 20% after 48 h of incubation . AccordiLow anticancer activity of anthocyanins and the phenolic acids-rich fraction of lingonberry fruits (F2), especially against colon adenocarcinoma cell line, was consistent with McDougall et al., who noted that anthocyanins-rich fraction was considerably less effective, and anticancer activity of lingonberries was determined in the proanthocyanidins-rich fraction . AngelovO-galactoside and cyanidin-3-O-glycosides and they can be detected in urine and serum [Proanthocyanidins are not absorbed and they enter the large intestine where they start to be metabolized to simple phenolic compounds. Therefore, they can express their effects in an unaltered state . Lingonbnd serum ,56,57. Lnd serum . Certainnd serum .Phenolic compounds can express antioxidant activity via multiple mechanisms such as direct scavenging of ROS, reducing the oxidative substances or stabilizing them, chelating metal ions, or modulation of endogenous antioxidant systems . All theBecause of strong correlations between proanthocyanidins, quercetin levels, and antioxidant or anticancer activities in different fractions, these compounds can be considered as the major contributors to the biological activity of lingonberries. This was confirmed by our novel findings on cancer cell viability reducing activity of standard substances of major phenolics of lingonberries, which revealed the strongest anticancer activity of quercetin and procyanidin A2, as well as by our previous study, which showed the highest antioxidant activities of standard substances of procyanidins C1 and A2 . It can The phytochemical analysis of crude extracts and phenolic fractions of lingonberries, by a validated analytical method with further evaluation of their free radical scavenging, reducing, and chelating activities, and anticancer activity against three cancer cell lines revealed large phytochemical diversity and highlighted predominant lingonberry phenolics. Out of them, quercetin and proanthocyanidins can be regarded as markers of antioxidant and anticancer activities of extracts of lingonberry leaves and fruits. These lingonberry compounds might be beneficial as promising chemo-active agents for alternative novel phytopreparations and nutraceutical products.


Here we employed genetic and pharmacological approaches that target CB1R, and found that its specific nullification in RPTCs preserves bone mass and remodeling both under normo- and hyper-glycemic conditions, and that its chronic blockade prevents the development of diabetes-induced bone loss. These protective effects of negatively targeting CB1R specifically in RPTCs were associated with its ability to modulate erythropoietin (EPO) synthesis, a hormone known to affect bone mass and remodeling. Our findings highlight a novel molecular mechanism by which CB1R in RPTCs remotely regulates skeletal homeostasis via a kidney-to-bone axis that involves EPO.The renal proximal tubule cells (RPTCs), well-known for maintaining glucose and mineral homeostasis, play a critical role in the regulation of kidney function and bone remodeling. Deterioration in RPTC function may therefore lead to the development of diabetic kidney disease (DKD) and osteoporosis. Previously, we have shown that the cannabinoid-1 receptor (CB The skeleton is not merely a solid protective frame that facilitates locomotion—it is also an intricate endocrine system that regulates numerous essential physiological functions such as energy and mineral metabolism ,2. IndeeRecent evidence shows that the hormonal regulation of bone mineral homeostasis is, however, much more complex and involves several systemic, kidney-specific, or bone-specific factors, such as sex hormones, insulin, cortisol, glucocorticoids, calcitonin, bone morphogenic proteins (BMPs), Klotho, erythropoietin (EPO), and numerous cytokines ,6,7,8. AType 1 diabetes (T1D) is associated with major microvascular  and macrovascular (atherosclerosis) complications. Additionally, T1D is associated with a low bone mineral density (BMD) and an increased risk of fracture, resulting from ablation of the insulin bone-anabolic effect, the toxic hyperglycemia, inflammation, oxidative stress, and superfluous urinary calcium excretion ,10,11. F1R) is vastly expressed in many cell types in the kidney [1R “tone”, which we recently found to promote kidney dysfunction, injury, inflammation, oxidative stress, mitochondrial fragmentation, and fibrosis by compromising the function of the RPTCs [1R was found to ameliorate these kidney abnormalities, thus preventing the development of DKD [For the past decade our research group has focused on elucidating the role of the endocannabinoid (eCB) system in several physiological/pathological functions. Our findings indicate that this endogenous system has crucial effects on the maintenance of both glucose and bone homeostasis; thus, deviation from this homeostasis can cause two major disorders: Diabetes and osteoporosis, suggesting that modulating the activity of the eCB system holds therapeutic promise for treating these disorders ,15,16,17e kidney . Among the RPTCs ,20,21,22t of DKD ,20.1R in the etiology of bone loss resulting from T1D. Here, we describe a novel pathway by which CB1R, specifically in RPTCs, remotely regulates bone mass and function under physiological and pathological conditions via the kidney-to-bone mediator EPO. The overall findings of this study may indicate that targeting renal CB1R activity and/or EPO may provide a promising therapeutic option for the treatment of T1D-induced bone loss.Considering all these findings, we set out to examine the contribution of renal CB1R agonists arachidonyl-2′-chloroethylamide (ACEA), noladin ether (NE), and anandamide (AEA), as well as the fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase (MAGL) inhibitor JZL-195 were purchased from Cayman Chemicals. The CB1R antagonists SLV-319 and JD5037 were purchased from Haoyuan Chemexpress Co., Ltd . Pertussis toxin (PTX) was purchased from Sigma–Aldrich/Merck .Streptozotocin (STZ), the CB1−/− mice were generated by crossing mice containing two loxP sites flanking the open reading frame of the CB1R with the iL1-sglt2-Cre line, which is specifically expressed in the RPTCs, as described previously [Cnr1 gene locus, G50, G51, and G53 primers were used as described previously [All experimental protocols used here were approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem . Animal studies are in compliance with the ARRIVE guidelines , and areeviously . Mice weeviously .1−/− mice and their littermate controls were administered five consecutive intraperitoneal (IP) injections of STZ . Two weeks after the first injection, blood glucose levels were measured using a glucometer , and animals with blood glucose levels greater than 250 mg/dL were considered diabetic and therefore were included in the study. Control groups for each strain were given 0.1 mol/L citrate buffer (pH 4.5). STZ-induced diabetic C57Bl/6J mice were treated daily with a global CB1R antagonist SLV-319 , or vehicle  for 16 weeks. Body weights and glucose levels were monitored weekly. To assess the in vivo effects on bone mineralization, newly mineralized bone was vitally labeled by fluorochrome calcein  that was injected IP at 15 mg/kg 4 and 1 d before euthanasia. At the age of 26 weeks, the mice were euthanized by a cervical dislocation under anesthesia. Once euthanized, trunk blood was collected for determining the biochemical parameters, kidneys were taken and kept at −80 °C, the femoral bones and L3 vertebrae were separated, cleaned, and fixed in 10% phosphate buffered formalin (pH 7.2) for 48 h, and then kept in 70% ethanol until further use.In studies requiring the induction of T1D, male 8-week-old C57Bl/6J or RPTC-CBTotal calcium (CA2), alkaline phosphatase (ALP2S), and phosphate (PHOS) were determined in serum or urine (as defined in the figures) using the Cobas C-111 chemistry analyzer . Serum osteocalcin levels were determined using an EIA kit . Mouse C-telopeptide of type I collagen (CTX-1) was measured in the same specimens using an EIA kit . Serum insulin levels were determined by ELISA .The skeletal phenotype was analyzed by a combinatorial µCT/histomorphometric system as described previously . BrieflyA three-point bending test was performed as reported previously . Briefly2/95% air. To test the effect of CB1R activation/blockade, cells were challenged with one of the following synthetic cannabinoids: 5 µM AEA, 10 µM ACEA, 2.5 µM NE, 250 nM JZL-195, or 100 nM JD5037 for 24 h. At the end of the incubation period, the cells were harvested for further analyses.Human immortalized RPTCs (HK-2) and the epithelial-like pig kidney cell line (LLCPK-1) were maintained in a low glucose DMEM  supplemented with 5% or 10% FCS, respectively. Cells were cultured at 37 °C in a humidified atmosphere of 5% COTotal mRNA from in vivo studies (kidney homogenate) and in vitro studies (HK-2 or LLCPK-1 cells) was extracted using Bio-Tri RNA lysis buffer  followed by DNase I treatment . RNA purity was validated spectrophotometrically by nanodrop 2000 , and reverse transcribed using the iScript cDNA kit . Real-time PCR analysis of 6 biological replicates in duplicates was performed using iTaq Universal SYBR Green Supermix  and the CFX connect ST system . The primers, used to assess the expression of various genes, were validated by blast sequencing, checked for specificity using the melting curve and Cq of the no template control (NTC) as well as optimized by defining the best annealing temperature, are listed in ®  and zirconium oxide beads . Protein concentrations were measured with the Pierce™ BCA Protein Assay kit . Cleared lysates were added with protein sample buffer, resolved by SDS-PAGE , and transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System . The blots were then washed with 1× TBS-T . Membranes were incubated for 1 h in 5% milk (in 1× TBS-T) to block unspecific binding, washed briefly, and incubated overnight at 4 °C with a primary antibody against EPO  at a dilution recommended by the supplier (1:500). EPO antibody specificity was assessed by using its positive (kidney and liver) and negative (muscle and brain) controls as described in the literature [Kidney and cell homogenates were prepared in a RIPA buffer  using BulletBlenderterature . The mem2O2 solution in MeOH. Unspecific antigens were blockaded by incubating sections for 1 h with 2.5% horse serum . For assessing the cellular structure, pancreas sections were stained with guinea pig anti-insulin . Endogenous peroxide was inhibited by incubating with a freshly prepared 3% Ht-test was used to determine differences between data sets comprised of two groups. Results in multiple groups were compared by ANOVA using Tukey’s multiple comparison test (GraphPad Prism v6 for Windows). Significance was set at p < 0.05.Values are expressed as the mean ± SEM. An unpaired two-tailed 1R in the regulation of skeletal remodeling and mass, the skeletal phenotype of mice lacking CB1R in RPTCs was compared to that of their littermate wild-type (WT) controls. Trabecular bone parameters were analyzed at the distal femoral metaphysis, a well-established site for assessing both catabolic and anabolic stimuli [1−/− mice were characterized by a high bone mass phenotype, manifested by a 17% increase in trabecular bone volume fraction (BV/TV), increased bone mineral density (BMD), and full femoral BV/TV  together with increased thickness of the cortical bone (Ct.Th.), without changes in the general diameter of the bone   I,K–M.1−/− mice is due to changes in bone formation or resorption, we next examined the presence and activity of osteoblasts and osteoclasts by using histomorphometric analyses. In fact, an enhanced mineral apposition rate  values in the diabetic WT group by utilizing µCT P. No cha animals M–P, sugg1−/− mice when compared either to their non-diabetic controls or to their WT littermate controls  and number (MS/BS), resulting in a reduction of BFR as well as reduced circulating osteocalcin levels in WT animals. No such effects, however, were found in the diabetic RPTC-CBcontrols A–D. In pcontrols E–G, coupcontrols H, PHOS , a globally acting CB1R blocker, attenuated the bone loss in an STZ-induced T1D model  with the CB1R activator, ACEA . As shown in 1R, suggesting a novel mechanism by which the EPO levels are regulated.Encouraged by the findings that genetic deletion of CBl tubule  is the il tubule ,34, whicl tubule ), we fir−/− mice A. To det1R activation or blockade on EPO levels in vitro. Interestingly, we found that activation of CB1R in two kidney cell lines, HK-2 and LLCPK-1, either by using direct agonists of CB1R  or by indirectly elevating eCB levels (using JZL-195), resulted in an elevation in the mRNA , coupled to the Gi/o subclass of G-proteins, we next evaluated the signal transduction connecting CB1R activation to EPO. To that end, we pretreated the cells with PTX, which inhibits the Gi-signaling pathway, prior to treating them with NE, and found that NE lost its ability to upregulate EPO protein expression  and/or fatty acid flux (obesity), and ameliorates diabetes- and obesity-induced albuminuria, fibrosis, and renal inflammation [1R located either in podocytes and/or RPTCs, since their specific nullification from these cells completely prevented the development of hyperglycemia- or lipotoxicity-induced renal dysfunction.CBiewed in ). Its efiewed in ,37,38, sammation ,22,39,401R, highly expressed in many cells within the kidney, is also upregulated in these conditions [1R system in regulating skeletal remodeling and mass in health and disease led us to test the hypothesis that a negative axis, involving CB1R in RPTCs, exists between kidney and bone. Indeed, nullification of CB1R from the RPTCs under normoglycemic conditions led to a high peak bone mass phenotype in mice, which can be attributed to increased BFR rather than reduced osteoclastogenesis, since enhanced osteoblast activity was measured in vivo in RPTC-CB1−/− mice. Osteocalcin, secreted almost exclusively by osteoblasts [1−/− mice, despite the fact that they had a high bone mass phenotype. The unchanged levels in the bone resorption marker CTX-1 [Since the kidney is a major source of eCBs, whose levels are elevated during diabetes and obesity ,41, and nditions ,45,46,47eoblasts ,49, was 1R in RPTCs significantly hindered T1D-induced bone loss in comparison with their diabetic WT controls. The reduction in bone mass observed in the diabetic WT animals, supported by previous findings in diabetic patients and mouse models [1R in RPTCs, suggest a novel therapeutic target. Indeed, similar findings were found in diabetic mice treated with SLV-319, which ameliorated hyperglycemia-induced bone loss without affecting systemic glucose homeostasis. These skeletal improvements are most likely related to the preserved renal function in these animals, as we reported previously [1R blockade was found to ameliorate bone morphological changes induced by chronic intermittent hypoxia in rats [1R blockade were age dependent [1R in RPTCs or that were treated with SLV-319.Next, we found that deleting CBe models ,52,53,54eviously , and fur in rats , as well in rats . Interesependent . Impaireependent ,59,60. A1R specifically in RPTCs significantly attenuated the increased osteoclast number without changing the CTX-1 levels. Comparable findings, demonstrating an insignificant effect of hyperglycemia on the serum levels of CTX-1 with an increased number of osteoclasts, were also reported by others [1R deletion or blockade, one should note the elevated circulating levels of PHOS and ALPL, which are known to be correlated with an increased bone turnover phenotype [Unlike the effect of hyperglycemia on osteoblasts, the current evidence that examined the role of T1D in affecting osteoclast number and function remains controversial. Our findings indicate that pharmacological blockade or genetic deletion of CBy others ,62; howey others . In supphenotype ,65,66,67henotype ,60,68, f1R [1R in influencing bone impairment during diabetes via affecting EPO levels. In accordance with renal hypoxia being a common feature of DKD [1−/− mice. An opposite effect, demonstrating an upregulation of renal EPO levels, was found in non-diabetic WT animals treated with the specific CB1R agonist ACEA. In fact, stimulating CB1R has been previously shown to induce hypoxia in the kidney and other tissues, whereas CB1R deletion/blockade ameliorated it [1R is a novel regulator of EPO synthesis. To further demonstrate a direct regulation of EPO by CB1R in RPTCs, we utilized two cell lines (from different sources: Human and pig), and showed that indeed the gene and protein levels of EPO are elevated following the activation of CB1R. Furthermore, indirectly activating CB1R, by using JZL-195, which inhibits FAAH and MAGL, the enzymes responsible for AEA and 2-AG degradation, respectively, resulted in a similar upregulation in EPO levels, an effect that was blocked by the CB1R antagonist JD5037. These effects were Gi-dependent, since NE did not affect the EPO levels in the presence of PTX. A previous study reported that AEA increased the EPO-induced stimulation of hematopoietic cell proliferation [2R. Therefore, the current study is the first to describe a direct relationship between CB1R and EPO.Among the various kidney-related bone modulators, recent evidence supports the critical role of EPO in regulating bone homeostasis either directly or systemically (reviewed in ). Since 1R ,20,22, ie of DKD ,70, and e of DKD ,72,73, wrated it ,55,74,75feration ; however1R increases the production and secretion of EPO to the serum, which in turn, may lead to bone loss. This hypothesis, reinforced by our findings, is based on the growing evidence that EPO negatively affects skeletal remodeling, and that its levels were found to be correlated with a low trabecular bone mass phenotype [Based on our current findings, it is reasonable to assume that activating CBhenotype ,77,78,79henotype . Followihenotype . Additiohenotype , which ihenotype . Howeverhenotype ,84. Our 1R in regulating bone remodeling and mass via EPO. However, it does not rule out the plausible roles of additional factors, such as 1,25(OH)2D3, Klotho, BMP-7, PTH, osteoprotegerin, sclerostin, and FGF-23, which influence bone mass and remodeling and could also play an important role in the skeletal phenotype described here. In fact, several studies reveal their contribution to diabetes-induced osteoporosis [1R system.The current study suggests a role for RPTC-CBoporosis ,89,90,911R. Nullification of CB1R from the RPTCs preserved bone mass under hyperglycemic conditions. This was achieved via affecting both osteoclastogenesis and bone formation. Although the exact mechanism underlying this phenomenon remains unknown, we suggest that EPO, generated by the kidney, is most likely involved, since its role in bone metabolism has been repeatedly demonstrated, and its levels are elevated during diabetes and are explicitly regulated by RPTC-CB1R. From a pharmacological point of view, the antagonism of CB1R in RPTCs has a therapeutic potential in treating diabetes-induced osteoporosis, and in improving the quality of life of patients suffering from diabetes.This work presents a possibly novel kidney-to-bone axis modulated via RPTC-CB


Epigenetic alterations, as a cancer hallmark, are associated with cancer initiation, progression and aggressiveness. Considering, however, that these alterations are reversible, drugs that target epigenetic machinery may have an inhibitory effect upon cancer treatment. The traditional drug discovery pathway is time-consuming and expensive, and thus, new and more effective strategies are required. Drug Repurposing (DR) comprises the discovery of a new medical indication for a drug that is approved for another indication, which has been recalled, that was not accepted or failed to prove efficacy. DR presents several advantages, mainly reduced resources, absence of the initial target discovery process and the reduced time necessary for the drug to be commercially available. There are numerous old drugs that are under study as repurposed epigenetic inhibitors which have demonstrated promising results in in vitro tumor models. Herein, we summarize the DR process and explore several repurposed drugs with different epigenetic targets that constitute promising candidates for cancer treatment, highlighting their mechanisms of action. Cancer is a disease that affects millions of citizens worldwide , being tS-adenosylmethionine (SAM), to the fifth carbon of the cytosine of CpG dinucleotide [Glutathione S-transferase pi 1 (GSTP1), involved in DNA protection, androgen receptor (AR), in prostate cancer [Both genetic and epigenetic mechanisms play an important role in malignant transformation, cancer initiation, tumor progression and prognosis . Epigenecleotide . Aberrane cancer ,5, estro3) [Alongside DNA methylation, histone modifications also play a role in carcinogenesis. Histones may endure posttranslational modifications at N-terminal tails, of which acetylation and methylation seem to be the most relevant . Histone3) . LSD1, a3) .Because epigenetic alterations are associated with cancer progression/aggressiveness, and considering that these alterations are reversible, drugs that target epigenetic enzymes may revert those alterations and contribute to the attenuation of the malignant phenotype .The traditional drug discovery pathway is costly, time consuming and has a low success rate. Considering these bottlenecks, new methodologies have been tested and the Drug Repurposing process has emerged as an interesting approach in cancer therapy .Drug Repurposing (DR) refers to the process of discovery of a new medical indication for a drug that was approved for another indication, removed from the market due to adverse events, was not accepted for the proposed indication or failed to prove efficacy ,12,13. DThere are several examples of drugs that were repurposed into new therapeutic approaches. Sildenafil is a drug that was originally developed for the treatment of coronary artery disease but which failed to pass on phase II clinical trials. One of the side effects verified upon treatment with Sildenafil was penile erection. Hence, this drug was repurposed by the FDA for the treatment of erectile dysfunction, in 1998 ,14,15. OIn Oncology, epigenetic alterations are becoming a therapeutic target. Food and Drug Administration (FDA) has approved two epigenetic modulators for cancer therapy that are repurposed drugs: 5-azacytidine and 5-aza-2′-deoxycytidine . These dDR is an important tool for novel, targeted therapies, and in this review, we will explore several repurposing drugs for epigenetic targets that might be promising candidates for Prostate Cancer (PCa) treatment.DNMT inhibitors (DNMTi) are the most studied epigenetic inhibitors. Presently, there are several drugs that can be repurposed for DNMTi .S-adenosyl-L-homocysteine (SAH) [p15 promoter demethylation. It was also shown to have nonepigenetic effects, causing reduced cell proliferation and cell cycle arrest at G0/G1 phase [RASSF1A promoter methylation and inducing re-expression [Chlorogenic acid is a coffee polyphenol that has been shown to inhibit DNMT1. Its inhibitory effect is due to a chemical transformation resulting in increased formation of ne (SAH) . Using bne (SAH) . In addiG1 phase ,42. FurtG1 phase  and in RG1 phase . MahaninG1 phase . In PCa pression ,31.WIF-1 [GSTP1 [ER, RARβ, p12 and p16 [RARβ, p21, p16 and APC gene expression in breast, bladder and cervical cancer cell lines, respectively [AR and p21 expression and inhibits the Epidermal Growth Factor Receptor (EGFR) bypass signaling pathway [The local anesthetic procaine is another interesting candidate for DR in cancer. It is a nonnucleoside inhibitor of DNMT1 and DNMT3A that binds to the binding pocket of the enzyme, disrupting the attachment of DNMTs to DNA . In breaWIF-1 , GSTP1 [1 [GSTP1 , ER, RAR and p16 . Hydrala and p16 ,44. Deng and p16  have shoectively ,25,26. Aectively  showed t pathway . Additio pathway .Finally, some antibiotics are also being studied. Mithramycin A has the potential to inhibit DNMT. Lin et al.  studied In PCa, HDAC enzymes are overexpressed, and due to the heterogeneity among subclasses, it is challenging to develop new drugs that target these epigenetic enzymes. Nonetheless, several approved drugs have been studied as potential HDAC inhibitors (HDACi) .Sp1 at RECK gene promoter [p21 and p27 expression, downregulation of cyclin D1 and cyclin E gene expression and cell cycle arrest [p21 gene expression. Pandey and colleagues also verified the effect of aspigenin in an in vivo model, in which the compound reduced class I HDAC activity, decreasing HDAC1 and HDAC3 protein levels, reducing tumor size, promoting apoptosis and upregulating p21 gene expression along with Bcl-2 downregulation [p21 gene expression and promoted cell death [Helminthosporium carbonum (HC)-toxin, a cyclic tetrapeptide derived from a plant, has been identified as HDAC inhibitor in different cell models. In breast cancer cell lines, HC-toxin inhibits HDAC activity and promotes nonepigenetic effects, specifically, cell proliferation inhibition, cell death and cell cycle arrest at G2/M phase [Pseudoceratina purpurea derivative Psammaplin A can be reduced to its monomers inside the cells due to the presence of disulfide bounds. These monomers, which have thiol groups, are key factors for the inhibition HDAC activity [p21 expression upregulation, inhibition of Rb, cyclins and cyclin-dependent kinase (CDK) gene expression, cell cycle arrest [2/M phase [Apicidin is a fungal metabolite that has been repurposed as an inhibitor of HDAC3, HDAC4 and HDAC8. Apicidin reduces HDAC3 and HDAC4 expression and activity, leading to increased histone H3 and H4 acetylation in endometrial and ovarian cancer cell lines ,77. In tpromoter . Furtherpromoter . In an ipromoter . Moreovepromoter ,47,78, ae arrest ,48. Furte arrest  demonstrgulation . Ginsengll death . Further/M phase . Additio/M phase . The spoactivity . Psammapactivity . It alsoactivity ,64. In aactivity , p21 expe arrest  and alsoe arrest  demonstr/M phase .p21 gene expression [1/G2 phase and increased apoptosis [p21, p27 and p57 expression in colon cancer cell lines [cyclin D1 gene expression [1 phase, increased expression of Bax gene and downregulated Bcl-2 gene expression and decreased phosphorylation of Akt and ERK proteins [The antimalarial drug artemisin has been repurposed as an HDAC1, HDAC2 and HDAC6 inhibitor. In breast cancer cell line MCF-7, Kumari et al.  demonstrpression . On the pression . Sodium poptosis ,66,67. Mpoptosis , downregpoptosis , increaspoptosis  and estrpoptosis , increaspoptosis  and uprell lines  Additionll lines , and affll lines . In addill lines ,74, incrll lines ,75 with ll lines , increaspression , cell cyproteins . There iIn recent years, HAT, HMT, HDM and BET inhibitors have gained interest from the scientific community; several drugs have shown promise as repurposed inhibitors of these histone modulators .Plumbago zeylanica has been repurposed as a noncompetitive p300 inhibitor. In a liver cancer cell line, plumbagin inhibited p300 HAT activity, hence preventing p53 acetylation; it also decreased histone H3 and H4 acetylation and showed nonepigenetic effects, specifically, enhancement of apoptosis [Several natural compounds have been investigated as repurposed histone modulators inhibitors. Anacardic acid, an extract from cashew nutshell, showed anticancer and anti-inflammatory properties. It was found to have HATi properties, specifically acting as a p300 and Tip60 inhibitor at nanomolar and micromolar levels . Sun andpoptosis . Sakane poptosis .Furthermore, clorgyline and pargyline, both monoamine oxidase (MAO) inhibitors, have been repurposed as LSD1 inhibitors for solid and liquid tumors. In 2013, Han et al. investigated the effect of clorgyline in leukemia, colon and bladder cancer cell lines. They demonstrated that clorgyline inhibits LSD1 activity and decreases histone H3, lysine 4 mono and di-methylation, thus promoting an open chromatin state and the re-expression of silenced genes . AdditioNitroxoline is an FDA-approved drug for the treatment of urinary infections which has been repurposed as BETi, with selectivity for BRD4. Nitroxoline occupies the acetylated lysine pocket of BRD4, preventing binding to acetylated lysine residues . TherefoAmong epigenetic targets, DNMT and HDAC enzymes are the most studied. In cancer, altered DNMT and HDAC expression are linked together, driving downregulation of tumor suppressor gene expression . Therefop53 expression through DNA hypomethylation [BRCA1, p53, p21). Moreover, the effect of resveratrol as a repurposed cancer drug was also investigated in clinical trials .Berberine is a natural compound used for the treatment of parasitic and fungal infections which has been repurposed as DNMT and HDAC dual inhibitor . Regardihylation . Moreovehylation . Additiohylation ,99,100. hylation , induceshylation ,102,110.hylation . Additiohylation ,104,110.hylation . In a brhylation ,107. Addhylation . Resverahylation  and induhylation , upregulThe previously cited studies demonstrate that old drugs can be reused for new clinical applications, thus broadening their previously intended application . This isHowever, most studies used a small range of drug concentrations in a limited number of cell lines, and mainly in 2D settings, thus failing to demonstrate efficacy in more complex models such as 3D culture and in vivo assays. In these 2D models, cells grow in synthetic plastic surfaces, which represents a highly reductionist model due to the loss of extracellular matrices (ECM), cell-cell communication, differentiation and polarization . Therefo


Conversely, early H2S-based interventions have proved beneficial in preventing adult-onset CVD in animal studies via reversing programming processes by so-called reprogramming. The focus of this review will first summarize the current knowledge on H2S implicated in cardiovascular programming. This will be followed by supporting evidence for the links between H2S signaling and underlying mechanisms of cardiovascular programming, such as oxidative stress, nitric oxide deficiency, dysregulated nutrient-sensing signals, activation of the renin–angiotensin system, and gut microbiota dysbiosis. It will also provide an overview from animal models regarding how H2S-based reprogramming interventions, such as precursors of H2S and H2S donors, may prevent CVD of developmental origins. A better understanding of cardiovascular programming and recent advances in H2S-based interventions might provide the answers to bring down the global burden of CVD.The cardiovascular system can be programmed by a diversity of early-life insults, leading to cardiovascular disease (CVD) in adulthood. This notion is now termed developmental origins of health and disease (DOHaD). Emerging evidence indicates hydrogen sulfide (H Cardiovascular disease (CVD) is the leading cause of death worldwide, accounting for almost one third of all global deaths . CVD is The fetal cardiovascular system is vulnerable to adverse early-life environmental insults . Develop2S), the third gasotransmitter, has emerged as a crucial regulator of cardiovascular homeostasis [2S exerts multifaceted biological functions, including vasodilatation, angiogenesis, antioxidant, anti-inflammation, mitochondria bioenergetics, and antiapoptosis [2S-releasing drugs have been considered as potential therapeutics for CVD [2S can be used as a reprogramming strategy in hypertension of developmental origins [2S has been shown to have beneficial effects on CVD [Hydrogen sulfide  and carbon monoxide (CO) [2S is known as a ubiquitous second messenger molecule with important functions in cardiovascular physiology [2S has been directly focused on incident CVD; however, there is a growing need to better understand the mechanisms and pathways of H2S signaling in CVD of developmental origins.Hal roles . In the ide (CO) . Currentysiology ,15. Much2S synthesis, including enzymatic pathway, nonenzymatic pathway, and bacteria origins. Three enzymes have been identified to enzymatically generate H2S, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (3MST) [l-cysteine is the principal substrate for both CBS and CSE to generate H2S. CBS and CSE can also produce H2S using other substrates. Homocysteine can be catalyzed by CBS to generate cystathionine, followed by CSE to produce l-cysteine. All of the above-mentioned H2S-generating enzymes are expressed in the heart and blood vessels [2S, is provided by cysteine aminotransferase (CAT) and D-amino acid oxidase (DAO). In the peroxisome, d-cysteine can be catabolized by DAO to generate H2S [2S can be nonenzymatically produced through thiosulfate, glucose, polysulfides, glutathione, and elemental sulfur.e (3MST) . CBS and vessels ,16. In arate H2S . Besides2S is coming from the gut microbiota. Approximately fifty percent of fecal H2S is derived from bacteria. In the gut, sulfate-reducing bacteria (SRB) obtain energy from the oxidation of organic compounds, reducing sulfate to H2S. Desulfovibrio account for 66% of all SRB in the human colon [2S by sulfite reduction, including species E. coli, Enterobacter, Salmonella, Klebsiella, Bacillus, Corynebacterium, Staphylococcus, and Rhodococcus [2S via sulfur oxidation. The SOB members include genera Acidithiobacillus, Bacillus, Paracoccus, Pseudomonas, and Xanthobacter. In the gut, a huge quantity of H2S is oxidized by colonocytes to thiosulfate. The existence of thiosulfate in cecal venous blood not only reflects the detoxification of H2S but also the recycling of H2S.Another source of Han colon . Other gdococcus . Convers2S can be scavenged and stored in the bound-sulfate and sulfane sulfur pools. Methylation and oxidation are two major mechanisms of H2S metabolism. H2S can be excreted in urine and flatus as free sulfate, free sulfide or thiosulfate.In the circulation and tissues, free H2S plays a crucial role in the pathogenesis of CVD. The first are reports on knockout mice lacking genes encoding for CSE, CBS, and 3-MST. CSE is the most relevant H2S-producing enzyme in the cardiovascular system. Mutant mice lacking CSE had decreased H2S levels in the serum, heart, vessels, and other tissues [2S-generating pathways were found in CVDs, including atherosclerosis [Multiple lines of evidence indicate that H tissues . CSE kno tissues ,21. CBS- tissues  and cere tissues . 3-MST k tissues . Second,clerosis , coronarclerosis , stroke clerosis , and perclerosis .2S [2S is able to S-sulfhydrate Kelch-like ECH associated protein 1 (Keap1), specificity protein-1 (SP-1), nuclear factor kappa-B (NF-κB) and interferon regulatory factor-1 (IRF-1) to regulate target gene transcription, which is crucial for the regulation of endothelial phenotypes, myocardial hypertrophy, mitochondrial biogenesis, oxidative stress, apoptosis and inflammation [Third, are studies of protein S-sulfhydration, a vital post-translational modification induced by H2S . S-sulfh2S . H2S is ammation .2S-releasing drugs have demonstrated considerable promise for beneficial effects against CVDs in various animal models [2S have been reported in the heart and vasculature. In the heart, the protective effects of H2S signaling was related to anti-inflammation, antiapoptosis, reduction of oxidative stress, and antifibrosis that leads to cardiac remodeling and functional improvements. In the vessels, H2S signaling can preserve endothelial NO synthase (eNOS)-derived NO production, while reducing oxidative stress, inflammation, fibrosis, and smooth muscle cell proliferation.Fourth, several Hl models ,8. As rel models , several2S can prime endothelial cells toward angiogenesis and contribute to relax vascular smooth muscle cells, and thereby reducing BP [2S homeostasis is involved in the pathogenesis of endothelial dysfunction, while the application of H2S-releasing drugs to increase endogenous H2S level can restore endothelial function and antagonize the progression of CVDs.Endothelial dysfunctions are associated with various CVDs, including hypertension, atherosclerosis, myocardial infarction, and the cardiovascular complications of diabetes. Hucing BP . A defic2S is a vasodilator. H2S has been reported to relax various blood vessels, such as the rat thoracic aorta, portal vein, and peripheral resistance vessels [2S deficiency in hypertension has been examined in various animal models of hypertension, including the spontaneously hypertensive rat (SHR) [2S on hypertension, as reviewed elsewhere [2S-based therapies could be used as reprogramming interventions perinatally to reduce the vulnerability to developing cardiovascular programming in offspring.Hypertension is a key risk factor for multiple CVDs. Like NO, H vessels ,29,30. Tat (SHR) , the renat (SHR) , Dahl saat (SHR) , and NO-at (SHR) . Converslsewhere . However2S level and vascular CSE expression/activity, suggesting disturbance of the vascular CSE/H2S pathway plays a role in the pathogenesis of atherosclerosis [2S has also been noted in diabetic animal models and diabetic patients [2S replacement displayed myocardial protection [2S production is diminished in these pathological conditions and H2S deficiency contributes to the progression of disease [ApoE knockout mice developed advanced atherosclerosis related to a decreased plasma Hclerosis . Additiopatients . Converspatients . In a raotection . Likewisotection . Summari disease .Important support for cardiovascular programming came from epidemiological reports following birth cohorts in the severe famines  ,43. ThesAlthough the influence of fetal life for future cardiovascular health has been evidenced by various epidemiologic human studies, it is almost impossible to test prospectively for critical developmental windows in humans. Moreover, nearly all cohort studies involve offspring who have not yet reached middle age and defined cardiovascular endpoints. Therefore, it is difficult to establish direct cause-and-effect relationships between particular environmental insults and later clinical cardiovascular outcomes in these cohort studies. Accordingly, it would be logical to use animal models to test our knowledge, for which a developmental window is decisive for cardiovascular programming, to identify how types of early life insults may program cardiovascular phenotypes, and what reprogramming intervention can be applied.We propose a schema for summarizing the links between early-life insults, fetal programming, and the programming processes in different organ systems that are involved in the developmental programming of CVD, which is presented in 2S signaling pathway and the abovementioned mechanisms. We will discuss each of these mechanisms in turn.Despite a wide range of early-life environmental factors related to CVD in later life, current evidence suggests that there may be common molecular mechanisms underlying cardiovascular programming. Although the complete mechanisms remain inconclusive, animal models have provided important information on particular pathways including oxidative stress, NO, RAS, nutrient-sensing signals, and gut microbiota dysbiosis ,5,6. RemThe role of oxidative stress in the onset and progression of atherosclerosis and CVD has been widely studied ,54. Oxid2S has an antioxidant property, by which it is able to scavenge ROS, increase antioxidant glutathione, and activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor for protection against oxidative stress [2S synthesis, has been reported to reprogram hypertension in animal models of developmental hypertension, including suramin-induced preeclampsia [In contrast, a reprogramming strategy aimed at the reduction of oxidative stress by the use of perinatal antioxidants has been applied in animal models to prevent adult disease of developmental origins . H2S hase stress ,10,11. Aclampsia , and preclampsia .2S might counterbalance oxidative stress to protect offspring against cardiovascular programming. Nevertheless, whether the anti-oxidative ability of H2S might be interconnected with other mechanisms to prevent CVD of developmental origins remains to be elucidated.All together, these observations reveal that HEndothelial dysfunction, mainly characterized by NO deficiency, is the initial event in the development of CVD . NO defiG-nitro-L-arginine-methyl ester  causes cardiovascular programming in adult offspring, characterized as endothelial dysfunction, hypertension, defect of carotid artery, and cardiac hypotrophy [Abundant evidence indicates that the impaired ADMA/NO pathway contributes to the pathogenesis of cardiovascular programming. First, ADMA competes with L-arginine to inhibit NO production  and its potrophy ,76. Thirpotrophy ,78,79.2S and NO can affect not only the generation of each other but also the further downstream signaling pathway [2S and NO display some similar functions like regulation of vascular tone, stimulation of endothelial cell angiogenesis and protection against cardiac injury [2S has been reported to increase NO bioavailability via activation of eNOS through the Akt pathway or calcium release [2S and NO.On the other hand, growing evidence supports H pathway . In the c injury . H2S has release ,82, enha release , decreas release , and red release . AlthougThe RAS is a major hormone cascade involved in the cardiovascular system . There aBoth pathways have been implicated in fetal programming ,88. The 2S and the downregulation of its producing enzymes were reported in hypertensive models with activation of the classical RAS [2S against hypertension coincided with downregulating RAS-related mRNA expression [2S contributing to the protection of CVD of developmental origins await further investigation.Low levels of Hical RAS ,93. Convpression , reducinpression , and supMaternal nutritional status governs fetal growth and development by means of nutrient-sensing signals. Imbalanced maternal nutrition can disturb nutrient-sensing signals, leading to fetal programming and adverse cardiometabolic outcomes . In the Our prior research revealed that AMPK activation prevents the development of hypertension programmed via regulation of nutrient-sensing signals in various models of developmental hypertension, including high-fat diet , high-frBesides, AMPK, SIRT1, and PGC-1α can mediate autophagy, a self-degradative process that promotes proteolytic degradation of cytosolic components at the lysosome . AMPK ca2S-associated cardiovascular beneficial effects [2S-induced cytoprotective effect [2S is associated with AMPK phosphorylation and alleviation of autophagy in a myocardial ischemia mice model [2S could inhibit the excessive autophagy of vascular endothelial cells by regulating the AMPK signaling pathway [2S can promote SIRT1 activity to mediate angiogenesis [2S against ischemia/reperfusion (I/R) injury are related to the activation of SIRT1/PGC-1α in a rat I/R model [2S and nutrient-sensing signals are implicated in CVD of developmental origins.AMPK and SIRT1 are considered as main mediators of H effects . Activate effect . The carce model . In anot pathway . Moreoveogenesis . Another/R model . These oThe gut bacteria can affect the control of the cardiovascular system via two pathways . First, 2S [Adverse environmental conditions occurring early in life can alter the microbial composition of the gut, leading to many adult diseases like CVD ,117. So 2S ,119,120.Lactobacillus casei, or postbiotics acetate during pregnancy and lactation can protect adult offspring against hypertension programmed by a variety of maternal insults [Conversely, approaches including probiotics, prebiotics, postbiotics , or microbial inhibitors that target specific pathways , have shown beneficial cardiovascular effects . Our pre insults ,123,124.2S has been reported to display BP-lowering effect [2S on cardiovascular programming. Recently, data obtained from our laboratory demonstrated that maternal NAC therapy that protected male SHR offspring against hypertension was linked to increased fecal concentrations of H2S and thiosulfate, augmentation of H2S-producing pathway in the kidneys, and alterations of gut microbiota [2S and also an index of the sulfide pool [2S might be a potential approach to prevent hypertension and deserves further evaluation. Another study reported that a high-fat diet caused hypertension in adult offspring and was associated with reduced plasma and fetal H2S levels, renal H2S-releasing activity, and a decrease in α-diversity in the gut microbiota [2S-generating enzymes in offspring kidneys as well as increased microbial richness and microbial diversity [Although gut bacteria-derived Hg effect , there icrobiota . As thioide pool , our rescrobiota . Conversiversity .2S and other important mechanisms involved in cardiovascular programming. Although there is emerging evidence for crosstalk between H2S and particular mechanisms related to CVD of developmental origins, more research is required to gain insight into how H2S may play an essential role in mediating other mechanisms, to develop a specific strategy to reduce their impact on developmental programming of CVD.Overall, these findings establish a close connection between H2S and other mechanisms implicated in cardiovascular reprogramming by H2S-based interventions to prevent developmental programming of cardiovascular disease is depicted in A summary of the links between H2S-based agents as potential therapeutics [2S donors [2S. Naturally occurring H2S donors derived from garlic and onions generate free H2S at a slower rate than sulfide salts [2S donors [2S-releasing profile better mimics physiological H2S production. However, the poor pharmacological properties of sulfide salts and naturally occurring H2S donors warrant the need for further development of novel, synthetic H2S donors like SG-1002 [2S donors  [2S donors  [2S donors [2S-donating agents are designed and tested in preclinical models of CVD, few of them have been examined in CVD of developmental origins.In the past decade, there has been heightened enthusiasm for the development Hapeutics ,8. SulfiS donors . Sulfidede salts . EarlierS donors , and its SG-1002 . Additio donors) , pH-cont donors) , and enzS donors , have be2S-based reprogramming interventions in animal models of cardiovascular programming, restricting interventions to critical periods during early development [Here, we show elopment ,139,140.G-nitro-l-arginine-methyl-ester (L-NAME) induced preeclampsia model [2S-based therapies have been reported in rats ranging from 12 week to 8 months of age, which is roughly equivalent to human ages from young to middle adulthood. Available H2S-based treatment modalities used as reprogramming interventions include l-cysteine, d-cysteine, NAC, sodium hydrosulfide (NaHS), and garlic.As shown in ia model , prenataia model , maternaia model , high-faia model , maternaia model ,138, andia model ,140. Theia model ,139,140,ia model ,138 and l-cysteine is a sulfur-containing amino acid. Besides, l-cysteine is a component of glutathione, a potent antioxidant in our body. As it is the substrate for H2S, l-cysteine supplementation is a way to produce endogenous H2S in experimental studies. Since H2S and glutathione are closely linked to BP regulation [l-cysteine has shown an antihypertensive effect [2S precursor, d-cysteine, is nutritionally antagonistic, and, hence, has received less attention [d-cysteine supplementation protects against ischemia/reperfusion injury in the kidney [gulation ,141, l-ce effect . NAC, a e effect ,144. Anottention . Althouge kidney , its effd- or l-cysteine between four and six weeks of age were protected against hypertension and kidney injury at 12 weeks old [d-cysteine pathway has an 80-fold greater H2S-releasing activity compared to the l-cysteine pathway [2S precursors in response to various insults that have been studied displayed protection against hypertension, there remains a lack of data regarding other cardiovascular benefits.We previously presented that high salt-treated SHRs supplemented with eeks old . Althoug pathway , our res pathway . As such pathway , prenata pathway , L-NAME- pathway , and mat pathway . Althoug2S) are the most widely used H2S donors to evaluate the therapeutic potential of exogenous H2S [2S has shown an antihypertensive effect in SHR [Inorganic sulfide salts such as sodium hydrosulfide (NaHS) and sodium sulfide  have shown to be safe and well tolerated in Phase I clinical trials [2S in vivo. Currently, various methods have been established for endogenous H2S measurement. Nevertheless, these methods have obvious limitations, especially in the clinical setting [2S measurements in clinical practice and ensure H2S-based therapy would only apply in the case of deficits.It stands to reason that early-life Hl trials , attenti setting . Therefo2S-based intervention for the right person, at the right time, for clinical applications. Little reliable information currently exists regarding the reprogramming effects of H2S-releasing drugs in human trials. Much of the preclinical work investigating the cardiovascular reprogramming actions of H2S has mainly studied hypertension. Since children with chronic kidney disease are at high risk for future CVD and they develop hypertension at early stages of CKD [2S-based interventions can prevent the transition of prehypertension to hypertension [Meanwhile, we are aware that a long road still lies ahead in determining the right dose of Hs of CKD , and thartension ,136, the2S-releasing drugs, while less attention has been paid to gut bacteria-derived H2S. Lacking in the literature is how microbiota-targeted therapies may alter SRB to produce gut-derived H2S and whether it is beneficial or harmful for cardiovascular health.Another important aspect is that significant progress has been made over the last few decades in H2S signaling pathway, has been linked to other common molecular mechanisms. What is the exact manner of cross-talk between these pathways? Are early-life interventions targeting other mechanisms  also able to mediate the H2S pathway to prevent CVD of developmental origins?Cardiovascular programming, apart from the H2S is a meaningfully pathogenetic link for the developmental origins of CVD. After all this tremendous growth in H2S-based interventions and greater understanding of cardiovascular programming, we expect that H2S-based reprogramming therapies will be applied in clinics to reduce the global burden of CVD.Finally, H


SFCOCz and SFCODPAC with charming aggregation-induced emission are expediently designed and prepared based on highly twisted strong electron-withdrawing acceptor (A) of sulfurafluorene (SF)-modified ketone (CO) and arylamine donor (D) in D1−A–D2 architecture by simple synthetic procedure in high yields. High photoluminescence quantum yields up to 73% and small singlet–triplet splitting of 0.03 eV; short exciton lifetimes are obtained in the resultant molecules. Strikingly, efficient non-doped and doped TADF organic light-emitting diodes (OLEDs) facilitated by these emitters show high luminance of 5,598 and 11,595 cd m−2, current efficiencies (CEs) of 16.8 and 35.6 cd/A, power efficiencies (PEs) of 9.1 and 29.8 lm/W, and external quantum efficiencies (EQEs) of 7.5 and 15.9%, respectively. This work furnishes a concrete instance in exploring efficient TADF emitter, which is highly conducive and encouraging in stimulating the development of TADF OLEDs with high brightness and excellent efficiencies simultaneously.The exploitation of thermally activated delayed fluorescence (TADF) emitters with aggregation-induced emission is highly prerequisite for the construction of highly efficient electroluminescent devices in materials science. Herein, two asymmetric TADF emitters of  EST) for efficient reverse intersystem crossing (RISC), but also due to their fundamental significance both in scientific investigations and technological applications of organic electronics  slight overlap of frontier molecular orbital (FMO) for maintaining high photoluminescence quantum yield (PLQY); (iii) short exciton lifetimes to eliminate the concentration-induced quenching effect; (iv) reduced intermolecular interactions to alleviate aggregation quenching processes; and (v) acceptable thermal stability for long-term device operation and ease synthesis procedure for mass productions. Nonetheless, most of the reported TADF materials suffer from serious aggregation caused quenching (ACQ) phenomenon that obviously hinders their practical applications  have been widely investigated not only because of their great potential in utilizing theoretically 100% internal quantum efficiency (IQE) through the back transfer of non-radiative triplet exactions (75%) into radiative singlet excitons conferred by small single–triplet energy splitting  is a fascinating optical phenomenon with greatly enhanced luminescent efficiency in solid state, which has been increasingly emerging as a promising candidate in organic electronics, bio-electronics, and photonics -modified ketone (CO) and arylamine of carbazole (Cz) or 9,9-diphenyl-9,10-dihydroacridine (DPAC). In this moiety, the D1−A–D2 can effectively render separated HOMO and LUMO distributions to guarantee a small ΔEST for the promotion of RISC process under thermal activation; moreover, the twisted and asymmetric molecular configuration can endow AIE and restrain intermolecular interaction of π-π stacking and/or aggregation to reduce ACQ in solid state  of ~73%, and efficient RISC rate and relatively short delayed fluorescence lifetime. High-performance non-doped and doped TADF OLEDs endowed by these emitters were fabricated, showing high luminance of 5,598 and 11,595 cd m−2 and external quantum efficiencies (EQEs) of 7.5 and 15.9%, respectively. The ease of synthetic route, excellent optoelectronics, and high device performance make the SFCO-based asymmetric TADF emitters promising candidates in practical applications, conferring a new paradigm for next-generation organoelectronics.Herein, to achieve the AIE-TADF materials, we designed and synthesized two emitters of itecture  through H-carbazol-9-yl)phenyl)methanone (SFCOCz) and dibenzothiophen-2-yl-yl)phenyl)methanone (SFCODPAC), were designed and synthesized in D1−A–D2 architecture through the direct connection of sulfurafluorene-tailored diphenyl ketone and donor of Cz (SFCOCz) or DPAC (SFCODPAC) by a conventional two-step procedure composed of Friedel–Crafts acylation and C–N coupling reaction  of 386 and 429°C as revealed by the thermogravimetric (TGA) measurements and melting temperature (Tm) of 197 and 251°C as measured by differential scanning calorimetry (DSC) analyses. The slightly higher Td and Tm of SFCODPAC than those of SFCOCz could be well-explained by its high molecular weight and rigid structure. In addition, the vacuum-evaporated thin films on glass substrates are amorphous and uniform with quite small root-mean-square roughness (RMS) of 0.307 and 0.173 nm for  and neat and doped films  from arylamine to CO , calculated by the onset edge of the absorption spectra, were 3.04 and 2.88 eV for SFCOCz and SFCODPAC, respectively. For the PL spectra, SFCOCz and SFCODPAC also show structureless ICT bands at ~450 and 492 nm with corresponding ΦPL of ~22 and 73%, respectively. Notably, the ΦPLs of SFCOCz and SFCODPAC in the solid state are up to ~3.7-fold that in the solution, indicating the obvious AIE characteristics. To further demonstrate the AIE properties of these two TADF molecules, the PL spectra in tetrahydrofuran (THF) with varied water fractions (fw) were performed. SFCOCz and SFCODPAC exhibit intense emissions as the formation of nanoaggregates upon injecting a large amount of poor solvent of H2O (fraction >80%) into THF solutions (SFCOCz (30 wt%) and SFCODPAC (30 wt%) doped films using 2,8-bis(diphenyl-phosphoryl)-dibenzothiophene (PPT) as the host material  doped films were 0.17 and 0.03 eV in the neat films, and 0.21 and 0.02 eV in the doped films, respectively, potentially enabling the process of RISC by thermal activation. The transient photoluminescence profiles were also performed to understand the photophysical process of SFCOCz and SFCODPAC  and a microsecond lifetime (τDF) of delayed emission  unit of Cz for SFCOCz and DPAC for SFCODPAC, while the LUMOs are mainly concentrated on CO and slightly located on the SF unit, showing an overlap extent (IH/L) of 32.2 and 10.9%, respectively  curves. According to the oxidation onset at 0.94 and 0.76 V, the HOMOs were evaluated to be −5.70 and −5.52 eV for SFCOCz and SFCODPAC, respectively. By means of optical bandgaps and HOMOs, the LUMOs were speculated to be −2.66 and −2.64 eV. To reveal the electron transition components of excited states in SFCOCz and SFCODPAC, the natural transition orbital (NTO) analyses were also carried out , and 65.7 and 22.2% on triplet state (IT) for SFCOCz and SFCODPAC, respectively. The overlap at the excited state should play an important role in guaranteeing high PLQY.As shown in 1−A–D2 molecules in constructing high-performance thermal-evaporated devices, the non-doped (TA-TB) and doped (TC-TD) TADF OLED of SFCOCz  and SFCODPAC  were successfully fabricated using the following configurations (3 (30 nm)/4,4′-bis[N-(1-naphthyl)-N-phenylamino]-1,1′-biphenyl (NPB) (50 nm)/1,3-bis(carbazol-9-yl)benzene (mCP) (5 nm)/EML (20 nm)/PPT (5 nm)/bathophenanthroline (BPhen) (5 nm)/LiF (1 nm)/Al (100 nm). In these devices, NPB and BPhen were hole- and electron-transporting layers, and mCP and PPT were exciton-blocking layers. TA-TD exhibited pure electroluminescence (EL) spectra inherited from their neat and doped films at different driving voltages, showing maximum emission peaks at 451, 482, 468, and 485 nm with corresponding Commission International de l'Eclairage (CIE) coordinates of , , , and , respectively  of 4.4 and 3.8 V, respectively. In addition to the decreased Von, TB and TD also demonstrated improved luminance up to 5,598 and 11,595 cd m−2 than those of TA and TC. The low Vons and high luminance of TB and TD should be attributed to its high HOMO and PLQYs of SFCODPAC for the promotion of hole injection from the adjacent layer and the generation of excitons. Not surprisingly, TADF OLEDs based on SFCODPAC show much better device efficiencies with current efficiencies (CEs) of 16.8 and 35.6 cd/A, power efficiencies (PEs) of 9.1 and 29.8 lm/W, and EQEs of 7.5 and 15.9% for TB and TD, respectively  and , respectively. Our work here provides a delicate molecular design strategy for the construction of asymmetric AIE-type TADF emitters, and clearly manifests the significant advance of the combined TADF and AIE features in exploiting high-performance organic emitters.In summary, we have succeeded in designing and developing two asymmetric TADF molecules of The datasets generated for this study can be found in the Cambridge Structural Database under the following identifiers: CCDC/1964730, CCDC/1964747.HuaL, YZ, YT, WH, and RC conceived the experiment. HuaL, YZ, YJ, and HuiL performed the characterizations of optoelectronic properties. YD, YT, and ML designed and fabricated the devices. QY and PL contributed the theoretical simulations. HuaL, YZ, YJ, and YT wrote the manuscript. All authors contributed to the scientific discussion.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.