Pnpla5 in rats causes a variety of phenotypic abnormalities. In this study, we further explored the effects of Pnpla5 knockout (KO) on male rats.Patatin-like phospholipase domain containing 5 (PNPLA5) is a newly-discovered lipase. Although the PNPLA family plays critical roles in diverse biological processes, the biological functions of PNPLA5 mostly unknown. We previously found that the deletion of Pnpla5 KO or wild-type (WT) male Sprague–Dawley rats were measured. The protein expression levels were also measured via western blotting and iTRAQ (isobaric tags for relative and absolute quantitation) analyses. No significant difference between Pnpla5 KO and WT rats, regarding body weight, testicular or epididymal tissue weight, or hormone levels, were found. However, the relative testicular tissue weight of the KO (Pnpla5−/−) rats was higher (P < 0.05) than that of WT rats. Significant increases in apoptotic cells numbers (P < 0.001) and BAX and Caspase-9 expression levels were observed in the testicular tissue of Pnpla5−/− rats. Moreover, iTRAQ analysis revealed that the levels of proteins involved in steroid metabolism and wound healing were significantly decreased in Pnpla5−/− rats.The body weight and testicular or epididymal tissue weight of three to six 3-month-old Pnpla5 knockout induced apoptosis in rat testes. We also ascertained that Pnpla5 plays an important role in lipid metabolism, wound healing, and affects reproductive organs negatively, providing new target genes and pathways that can be analyzed to unravel the biological function of Pnpla5.This study revealed that The online version contains supplementary material available at 10.1186/s12864-022-08835-8. Pnpla5 remain elusive. Although most tissues of rats [Pnpla5 is abundant in the skin, testes, epididymis, uterus, and ovaries [Pnpla5 is highly expressed in the lungs, epididymis and brown adipose tissues [Pnpla5 is highly correlated with that of Pnpla3, which located upstream of the Pnpla5 gene in the mouse genome. Both these genes are inhibited by fasting, up-regulated in the process of adipocyte differentiation and highly activated in the liver of obese (ob/ob) mice [Pnpla5 mRNA expression level increases in Pnpla3 KO mice, and it can be up-regulated to reach a level that equates untampered Pnpla3 level [Pnpla5 partially compensate for the biological deficiencies caused by the absence of Pnpla3.The patatin-like phospholipase domain-containing (PNPLA) protein family is a newly discovered type of lipases . Ten PNP of rats , mice, a of rats  contain  ovaries , while i tissues . The expob) mice . FurtherPnpla5 decreases the cellular TG content [Pnpla5 is associated with low density lipoprotein cholesterol (LDL-C) [Pnpla5 KO (Pnpla5−/−) Sprague–Dawley (SD) rats [Pnpla5−/− rats exhibited abnormal bleeding [Pnpla5 not only plays a key role in lipid metabolism, but also affects the fertility of male rats. To further explore the biological functions of Pnpla5, the body weight, testicular and epididymal tissue weight, and reproductive hormone levels of Pnpla5−/− male rats were measured. We detected germ cell apoptosis in rat testicular tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and western blotting analyses. We also performed iTRAQ (Isobaric tags for relative and absolute quantitation) proteomic analysis of testes to identify target genes and pathways that may be regulated by Pnpla5.PNPLA5 exhibits triglyceride (TG) hydrolase activity , 7, furt content . Whole-e (LDL-C) . In our SD) rats  and founbleeding  and redubleeding . These fPnpla5 plays an important role in lipid metabolism, and its deletion causes disorders of lipid metabolism in rats [Pnpla5 knockout significantly reduce the fertility of male rats and the curvilinear velocity of their sperm [. Therefore, we assessed the body and reproductive organs (testis and epididymis) weight, and serum hormone levels of Pnpla5−/− and WT (Pnpla5/+)+ male rats  in their body weight , but the relative epididymal weights were not significantly different . In addition, the expression levels of pro-apoptotic genes like Bax, Caspase 9 increased while those anti-apoptotic genes, such as Bcl2, decreased , and no significant differences were found  quantitative proteomic analyses to compare Pnpla5−/− with Pnpla5/++ rat testes. With false discovery rate (FDR) < 1.0% and specific peptides identified per protein ≥ 2, a total of 5118 proteins were identified using the UniProt rat protein database. Proteins that had an absolute value of log2 of KO/WT ≥ 0.26 and P-values < 0.05 (via t-test) were identified as differentially expressed proteins (DEPs). Forty nonredundant DEPs were identified in Pnpla5−/− rat testis , Pon1 (Paraoxonase 1), Hsd17b3 (Hydroxysteroid (17-beta) dehydrogenase 3), Akr1c19  and Hsd3b3  are involved in these steroid metabolic processes, and are all down-regulated in Pnpla5−/− rat testis  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed. The GO enrichment analysis Fig.  showed t−/− rats , indicatPnpla5−/− rats [Dcn (Decorin), Fmod (Fibromodulin), F10 (Coagulation factor X), Eng (Endoglin), C6 (Complement C6), Apoa2, Kng1 (Kininogen 1), and Kng2 (Kininogen 2). Dcn, Fmod, Kng2, Kng1, F10, and Eng were enriched in the wound healing GO term and C6, Apoa2, Kng1, and Kng2 were enriched in the acute inflammatory response GO term. Moreover, C6/Kng1/Kng2 and F10 were also enriched in the complement and coagulation cascades pathways  were significantly enriched. This is consistent with a previous report that Pnpla5−/− rats have abnormal serum TC, TG, HDL-C and LDL-C levels, indicating that Pnpla5 may play an important role in protein-lipid complex formation.The body and reproductive organs weight and serum hormone levels of ups Fig. , despitePnpla5-/- rats. Dcn, Fmod, Kng2, Kng1, F10, C6, Apoa2, and Eng were enriched in the wound healing and acute inflammatory response GO terms, and may contribute to the uncontrollable bleeding and longer blood coagulation time observed in Pnpla5-/- rats [F10, C6, Kng1, and Kng2 were also enriched in the complement and coagulation cascades pathway by KEGG enrichment. The down-regulation of F10/C6 directly blocks MAC formation [Pnpla5-/- rats may also suffer from recurrent bacterial infections.Quantitative proteomic analysis revealed several genes and potential pathways that may be involved in the molecular mechanisms underlying abnormal bleeding in -/- rats . Notablyormation , which iormation . TherefoPnpla5−/− male rats, indicative of reduced fertility and sperm motility, and supported by the disarranged histological structure of Pnpla5−/− male rats testes [Pnpla5 knockout may be one of the reasons for abnormalities in testicular tissue.Previous reports noted the decreased pregnancy rates in female rats that mated with s testes . In thiss testes . TherefoPnpla5−/− rats were used as the animal model to study the biological functions of Pnpla5. The data showed that this gene’s deletion in rats caused no significant difference in body, testicular or epididymal tissue weight, or hormones levels. However, Pnpla5 KO significantly increases testicular apoptosis level, indicating that the deletion of Pnpla5 adversely affects the reproductive organs. Moreover, iTRAQ proteomic analysis revealed that DEPs, involved in steroid metabolism and wound healing, were significantly decreased in Pnpla5−/− rats. Our study sheds new light, but does not fully address, the underlying mechanisms of phenotypic abnormalities induced by Pnpla5 knockout. Several questions remain unanswered at present. The fact that, Pnpla5 deletion had no effect on the TG content of testes, yet increased the TG serum levels, is a discrepancy that needs to be resolved in the future. Much work is yet to be done to fully characterize the biological functions of the Pnpla5 gene.In summary, Pnpla5 KO SD rats used in this study were obtained as previously described [The escribed . All ratPnpla5−/− and Pnpla5/++ male rats. Whole blood was centrifuged at 4000 × g for 10 min at room temperature to obtain the serum. Then serum testosterone, follicle-stimulating hormone, and luteinizing hormone levels were measured using a Hitachi 7080 Biochemical Automatic Analyzer .After pentobarbital sodium  anesthesia , blood samples were collected from the orbital of Gapdh was used as a control, and the relative gene expression level was calculated using the 2 − ΔΔCt method. The primers used are listed in Table STotal RNA was extracted from testes of rats using TRIzol , and reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis kit . qPCR was performed using TB Green® Premix Ex Taq II  following the manufacturer’s instruction. Pnpla5−/− and Pnpla5/++ testes were measured by a Tissue TG Content Assay Kit (Cat. No. E1013), following the manufacturer’s instructions.The TG content in the For western blotting, the tissues were homogenized, and the supernatants were collected after centrifugation. Total protein concentration was quantified using a BCA kit . Equal amounts of total protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis  and transferred to a polyvinylidene difluoride membrane . After blocking with 5% non-fat milk at 4 °C overnight, the membrane was incubated with primary antibodies at 4 °C overnight, washed three times with Tris-buffered saline with tween 20 (TBST) buffer , and then incubated with secondary antibodies at room temperature for 2 h. The membrane was washed three times with TBST buffer, and the blots were stained using an enhanced chemiluminescence (ECL) method on a Tanon-5200 Chemiluminescent Imaging System and ImageJ . Immunoblotting used the following primary antibodies: BAX , BCL2 , anti-caspase 9 , and β-actin antibody . We used anti-rabbit IgG  as the secondary antibody.Fixed rat testis was embedded in paraffin in vertical position. Then embedded testis-tissue was sliced with a microtome into 5‐7 µm thick sections and adhered to the slides. After incubated in a xylene bath and rehydration, the slides were stained with hematoxylin and eosin (HE). These HE stained slides were used for morphological analysis of the testicular structure.Pnpla5−/− and Pnpla5/++ rats were fixed in 4% formaldehyde for 24 h and embedded in paraffin. Then, 5‐7 μm paraffin sections were adhered to the slides. These section slides were incubated in a xylene bath, at room temperature, for 10 min. The slides were then rehydrated by being transferred through a graded ethanol series: 3 min in 90, 80, and 70% ethanol, each, followed by 3 min in double-distilled water. The sections were covered by pipetted proteinase K solution (20 mg/mL) and incubated for 15 min at room temperature. Thereafter, the slides underwent two 5-min PBS washing cycles. The sections were then covered with 50 µL TUNEL reaction buffer and converter-POD at 37 °C, for 1 h and 30 min, respectively, as well as 50 µL IDAB substrate for 10 min at room temperature. Thereafter, the slides were washed with PBS and counterstained with hematoxylin for 10 min. Then stained slides were then subjected to a decreasing alcohol series. After incubation for 20 min in xylene, the slides were mounted. Tissue sections were examined by microscopy. At least ten fields were randomly selected for each sample, the number of TUNEL-positive cells in each field was counted, and the average number of apoptotic cells in each sample was calculated.To evaluate the status of the testis cells, we preformed TUNEL assays, using the In Situ Cell Death Detection Kit . Briefly, testicular tissue samples from Pnpla5/++ andPnpla5−/− were analyzed. The rats were euthanized by cervical dislocation under deep anesthesia, and the testes were recovered and stored in liquid nitrogen. Total protein was isolated as previously described [Pnpla5−/− and Pnpla5/++testicular tissues, respectively. The labeled samples were then mixed and vacuum-dried.For the proteomics quantitative analysis, two groups of 3-month-old rats (n = 3), escribed . ProteinP < 0.05 and absolute value of log2 of KO/WT ≥ 0.26 were considered DEPs.The dried peptides were reconstituted in 10 µL of 0.1% formic acid and loaded onto a low-pH reversed-phase C18 column , interfaced with an Orbitrap Q-Exactive-plus . Three independent biological replicates were performed, and the resulting peptides were independently analyzed using LC–MS/MS. The raw data files, produced by mass spectrometry (MS) and protein quantification, were identified using Mascot (Version 2.5.1) search against the rat protein database (downloaded from UniProt). The parameters used for database searching were as follows: trypsin selected as the enzyme, a maximum of two missed cleavages were allowed, precursor mass tolerance was set to 10 ppm, fragment mass tolerance was set to 0.05 KDa, carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and acetyl at the N-terminal and lysine side chains were allowed as dynamic modifications. The FDR was set to 1% for protein identification. Proteins with P < 0.05 were considered significantly enriched by differential expressed genes. KEGG pathways with corrected P < 0.05 were considered significantly enriched. GO and KEGG enrichment results were visualized using the ggplot2 (version 3.3.5), pathview (version 3.1.4) [To better understand the biological functions of DEPs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the clusterProfiler (version 4.2.1) R package . GO termn 3.1.4) , and ggpP values less than 0.05, 0.01, and 0.001 are indicated by *, **, and ***, respectively. Each rat served as an experimental unit.All data are presented as means ± standard deviation (SD). Data from the two groups were compared using an unpaired t-test. Data were analyzed using GraphPad Prism version 8 . Additionalfile 1: Figure S1. The original western blotting gels of Figure 2C. Figure S2. Triglyceride content of Pnpla5+/+ and Pnpla5-/-rat testes. Figure S3. The Nlrp3 expression level in testes.Additionalfile 2: Table S1. GO enrichment result.Additionalfile 3: Table S2. KEGG enrichment result.Additionalfile 4: Table S3. qPCR primers.


Testicular blood flow parameters (TBF), testicular volume (TV) and echogenicity (TE), nitric oxide (NO), seminal alanine aminotransferase (ALT) and catalase (CAT) activities, total antioxidant capacity (TAC), malondialdehyde (MDA) content, and semen quality traits were all measured. The examinations started a week before (W-1), on the first supplementation day (W0), and weekly for eight consecutive weeks (W1-W8). There were marked (P < 0.05) increases in TBF (W3-W6) and TV, and a decrease in TE (W3-W5) in the CoQ10 group compared to the CON group. Similarly, testosterone (T) and NO levels (W3-W5) in the CoQ10 group were higher (P < 0.05) than those of the control group. The CoQ10 group demonstrated significant (P < 0.05) increases in seminal CAT (W4-W8) and TAC (W2-W6) activities and decreases in ALT (W4-W7) activity and MDA (W5-W8) concentration as compared to the control group. The CoQ10 group showed improvements (P < 0.05) at W3-W6 for sperm progressive motility, viability, and normal morphology and at W6-W8 for sperm concentration. In conclusion, oral CoQ10 supplementation improved testicular hemodynamics, testosterone production, semen quality, and antioxidant capacity in goat bucks during summer heat stress conditions.Oxidative stress (OS) is brought on by heat stress (HS), which weakens antioxidant defense and initiates OS. Since mitochondria are the primary source of reactive oxygen species (ROS), HS-mediated OS may be lessened by targeting mitochondria with particular antioxidants. The purpose of this study was to investigate the effect of oral coenzyme Q10 (CoQ10) supplementation on the reproductive performance of goat bucks under HS conditions. Ten mature bucks were randomly separated into two groups and housed in an environment with a high-temperature humidity index . The first group ( Recently, there have been raising concerns regarding the global warming issue, which could affect livestock production and reproduction  and nitrogen species (RNS)] during mitochondrial oxidative phosphorylation processes and the loss of compensatory antioxidant defenses and subsequent oxidative stress (OS)  in its actions against ROS attacks  synthesis with a subsequent decrease in testicular blood perfusion. Several studies  and 1.25 kg/head/day of green roughage. The feed ingredients based on the manufacturer were yellow corn, wheat bran, cotton cake, sunflower cake, soybean meal, gluten feed of 16% and 22%, molasses, lime, NaCl, mineral salts, vitamins, and anti-mycotoxins; while, the feed chemical compositions were crude protein (18%), fat (2%), ash (9%), fiber (15%), and total digestible nutrients (65%). Fresh water and mineral licks were available for the bucks ad libitum. Periodical vaccination and deworming protocols were implied, following the instructions of the general authority of veterinary services in Egypt.Ten Baladi goat bucks, with average body weight and age , were used in this study. These bucks were thoroughly examined before being used in the experimental procedures to exclude any buck with cardiovascular and andrological problems suck as orchitis, based on general examination of the vital signs and Doppler ultrasound scanning of the reproductive system. They were housed in a paddock throughout the experimental time points, with the availability of a 20 mTemperature humidity index (THI) was followed to assess whether the examined bucks were under heat stress conditions or not  received the programmed daily diet only , while the other group  received the programmed daily diet plus oral administration of coenzyme Q10 at a dose of 3 mg/kg BW  for six consecutive weeks (W1-W6). The CoQ10 dose was designed according to a previous study on goats . Bucks were divided into two equal groups; the first group , equipped with a 5–7.5 MHz linear array probe, was used for the assessment of the testicular hemodynamics (TH). Before the ultrasound examination, the animals were controlled without a tranquilizer to eliminate its impact on TH, and the scrotal hair on both sides of the testes and the spermatic cord was clean-shaven. Also, the Doppler sets including gate (0.5 mm), filter (50 Hz), and the angle between the Doppler beam and the longitudinal axis of the supra-testicular (STA) artery (< 60°) were adjusted and kept constant during the study . All samples were centrifuged at 3000 rpm for 15 min, and then the harvested plasma was stored (− 20 ℃) until further assessment. Species-specific T, FSH, LH, and E2 were assayed from plasma samples by commercial ELISA  kits with intra- and inter-assays variation coefficients were ≤ 10 and ≤ 12% for all measured hormones. For NO measuring, 100 µL of serum samples were mixed with equal volumes of Griess reagent [N-(l-naphthyl) ethylenediamine and sulphanilamide] and incubated for 18–20 min at room temperature. The optical density was recorded using a spectrophotometer (543 nm wavelength) and the NO (µM/L) concentration was calculated . The collected samples were transferred immediately to the laboratory and held in a water bath (35 °C) for further evaluation. Each semen sample was divided into two parts, the first portion for sperm microscopical evaluation and the second one for biochemical evaluation of TAC, CAT, ALT, and MDA. To exclude the negative effects of long-time sexual rest, the semen quality data regarding the week before CoQ10 supplementation (W-1) was discarded  was recorded using the improved Neubauer hemocytometer , viability  and normal morphology  were examined subjectively using a heated-stage (38 °C) light microscope , while, SCC ] semen sample was loaded on a pre-warmed glass slide (37 °C) and cover-slipped. The spermatozoa that had forward progressive motility were recorded and expressed in percent. The sperm viability and normal morphology percentages were examined employing the eosin-nigrosin staining , semen samples were centrifuged (4 °C) at 2000 g for 15 min, followed by storage of the harvested SP at -20 °C till later measurement. To exclude the inter-assay variations, all the biochemical evaluations (W0-W8) were performed on the same day after the end of the study. Colormetrically using a spectrophotometer, commercial research kits were used for assaying the levels and activities of TAC (mM/L), CAT (U/L), MDA (mM/mL) , and ALT  specifically at 510, 520, 534, and 505 nm wavelengths, respectively, following the manufacturer’s instructions  reacting with malondialdehyde in an acid medium for 30 min at 95 °C to produce a TBA product (pink product). The absorbance of the product was evaluated at 534 nm. The estimation was done according to the manufacturer’s instructions for the commercial kit following the principle described by Satoh  and Ohka2O2). The antioxidants in the sample eliminate a certain amount of the supplied H2O2. The residuum H2O2 is estimated colorimetrically by an enzymatic reaction which involves the transformation of 3,5, dichloro –2– hydroxy benzenesulfonate to a colored product that is read against distilled water at 505 nm. TAC was estimated using commercial test kits following the principle described by Koracevic et al. . The Statistical Package for Social Sciences  was used for all statistical analyses.Firstly, the raw data were checked for normality using the Shapiro–Wilk test. The differences between the right and left testis regarding the Doppler findings were non-significant; therefore, the data of both testes were pooled and the means in each time point were presented. The biological repetition was three times for ultrasound measurements, two times for semen quality, hormonal and biochemical assessments. The differences between control and CoQ10 groups (treatment effect), in terms of testicular hemodynamics values , circulating hormones , NO, semen quality traits , seminal oxidative biomarkers , and ALT activity throughout the studied time points (time effect) and combined treatment time effect were analyzed using repeated-measures ANOVA test, followed by Tukey 3) elevated significantly (P < 0.05) from W 3 (53.24 ± 2.74) till W 5 (50.65 ± 2.55) as shown in  difference in the TV values, while both TE and pixel heterogeneity (PH) means were affected by the treatment by declination from W 3  till W 5 , while the time effect did not significantly affect both parameters compared to the CON group  differences in the values of RI, PI, PSV, and EDV; while the time effect had significant (P < 0.01) differences in RI, PI, PSV, and CA without affecting the EDV. However, the treatment*week interaction had a non-significant effect on all Doppler values. Both Doppler indices (RI and PI) decreased (P < 0.001) from W3 (0.62 ± 0.02 and 0.71 ± 0.02) till W 6 (0.49 ± 0.02 and 0.61 ± 0.02). Concurrently, both PSV and CA markedly increased (P < 0.001) from W3 (19.45 ± 0.05 and 1008.3 ± 6.32) till W6 , respectively.The CoQ10 supplementation affected the main testicular artery vascularity as shown in Table P < 0.01) in the CoQ10 group compared to the CON group  from W3 (4.02 ± 0.14 and 56.25 ± 0.73) till W5 (4.21 ± 0.02 and 61.21 ± 0.08), respectively. Furthermore, the time*treatment interaction had significant differences in T (P < 0.01) and NO (P < 0.05) levels.The CoQ10 treatment and time*treatment interaction effects (CoQ10 vs CON) had a non-significant difference in the plasma levels of E2, FSH, and LH Fig. . Moreoveoup Fig. . The T (P < 0.01) the percentages of SPM, SV, and NS starting at W3 till W6. There was a carrying-over effect of treatment on SCC till W8 compared to the CON group.The data showing the effect of supplemental dietary CoQ10 on the semen characteristics of heat-stressed goat bucks  is presented in Table P < 0.01) effects of treatment (CoQ10 vs CON) and time on the values of MDA and TAC, and activities of CAT and ALT. There were marked increases in the levels of TAC (W2-W6) and CAT (W4-W8) activity, and decreases in the values of MDA (W5-W8) and activity of ALT (W4-W7) in the CoQ10 group compared to the CON group.The seminal plasma levels of MDA and TAC, and activities of CAT and ALT enzymes (CoQ10 vs CON) studied at different time points (W0-W8) under heat stress conditions are presented in Table Environmental heat stress deteriorates animal productivity and fertility potential  (Leal et al. 9 (Daudu In the present study, there were remarkable improvements in semen quality traits at W3-W6 for sperm progressive motility, membrane integrity, and normal morphology, and at W6-W8 for SCC in the CoQ10 group versus the control group. Since subjective motility evaluation herein might have a wide error range among the examined animals and time points; a further study is recommended using advanced semen quality assessment techniques. Plenty of reports on roosters (Sharideh et al.  9 Daudu . Thirdly 9 Daudu . Therefo 9 Daudu  exacerba 9 Daudu . A recen 9 Daudu . Based oOral Supplementation of CoQ10 during the summer season ameliorates the adverse effects of heat stress on the testicular hemodynamics and volume, testosterone production and sperm quality, and seminal antioxidant capacity which could improve goat breeding performance. Further studies are needed to investigate the role of CoQ10 supplementation in different male reproductive performance aspects such as sexual activity, more advanced semen quality techniques, and fertility potential.


Vascular diseases are one of the most common causes of death and morbidity. Lower extremity artery disease (LEAD), abdominal aortic aneurysm (AAA) and chronic venous disease (CVD) belong to this group of conditions and exhibit various presentations and courses; thus, there is an urgent need for revealing new biomarkers for monitoring and potential treatment. Next-generation sequencing of mRNA allows rapid and detailed transcriptome analysis, allowing us to pinpoint the most pronounced differences between the mRNA expression profiles of vascular disease patients. Comparison of expression data of 519 DNA-repair-related genes obtained from mRNA next-generation sequencing revealed significant transcriptomic marks characterizing AAA, CVD and LEAD. Statistical, gene set enrichment analysis (GSEA), gene ontology (GO) and literature analyses were applied and highlighted many DNA repair and accompanying processes, such as cohesin functions, oxidative stress, homologous recombination, ubiquitin turnover, chromatin remodelling and DNA double-strand break repair. Surprisingly, obtained data suggest the contribution of genes engaged in the regulatory function of DNA repair as a key component that could be used to distinguish between analyzed conditions. DNA repair–related genes depicted in the presented study as dysregulated in AAA, CVD and LEAD could be utilized in the design of new biomarkers or therapies associated with these diseases. The global burden of disease-related mortality is shaped mainly by the prevalence of noncommunicable diseases like cancer, diabetes mellitus, cardiovascular and chronic respiratory diseases. Within this set of conditions, those related to the cardiovascular system are one of the most common causes of death and morbidity. Moreover, they will exhibit a constant trend toward being more prevalent in future decades ,2,3.Peripheral arterial disease (PAD), along with coronary heart disease and stroke, is a pronounced cause of demise and debilitation due to atherosclerotic complications . Lower eAbdominal aortic aneurysm (AAA) is most commonly defined by the maximum diameter of the abdominal aorta in excess of 3 cm in either anterior–posterior or transverse planes or, alternatively, as a focal dilation at least 1.5 times larger than the diameter of the normal adjacent arterial segment . The preThe main characteristics of chronic venous disease (CVD) include chronic morphological and functional abnormalities of the venous system caused by venous hypertension, venous valve incompetency and reflux in veins of the lower limbs, leading to venous wall remodelling and vascular inflammation. The clinical presentation varies from telangiectasia and varicose veins to much more severe complications like leg edema, skin changes and ulcers ,10,11,12It is widely accepted that oxidative stress and resulting DNA damage is present in various cardiovascular conditions. In atherosclerosis, DNA damage and repair is an important element of disease onset and progression ,14,15. IBER could be utilized in therapies reducing ischemic injury, where enhanced mitochondrial DNA repair could have a benevolent effect on the state of the human heart muscle and cerebral vasculature in stroke models ,21.The presented research is a part of a larger project regarding the application of next-generation sequencing in the identification of the transcriptomic signatures of three vascular diseases: LEAD , CVD 2323 and AAp < 0.05) dissimilarities in age and body mass index (BMI)  . While Lhttps://www.genenames.org/) (accessed on 15 June 2021) was queried with the term “DNA repair”, yielding 449 hits. To provide further details, the obtained gene list was compared with the Human DNA Repair Genes (https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html) (accessed on 17 June 2021), and all genes not presented in the HUGO set were added. Moreover, the gene list was supplemented with the remaining ALKB homologs (ALKBH1-8 and FTO) due to their established role in nucleic acid modification and repair [https://www.genecards.org) (accessed on 18 June 2021), the final list of genes submitted to further analysis had 519 positions  methods were used to assess the differential expression analysis of the set of 519 selected genes in pairwise comparisons between studied groups . To supplement pairwise analysis, pooled group comparisons were also conducted. In the first instance, the transcriptomic landscape of genes in a pooled group including LEAD, AAA and CVD subjects was compared with gene expression in control group, to reveal a general scheme of DNA damage metabolism gene expression in vascular pathogenesis. The second association was performed between gene expression in a pooled group containing LEAD and AAA subjects, representing artery-related diseases and CVD patients, to pinpoint differences in DNA repair-associated pathological processes ongoing in arteries compared with venous systems.p values generated for each comparison  and fold change > 1.2 (for upregulated genes) or fold change < 0.8 (for downregulated genes) were selected, while genes indicated by UVE-PLS as informative, with a minimum reliable score equal to 5, were considered. Both methods gave separate sets of genes in each of the comparisons . Correspondingly, there were groups of genes with less pronounced differential expression, common for three comparisons : PNKP, DEK, POLE, SMC3, UPF1, WHSC1 (NDS2), XRCC6 (Ku60), POLE4, and for two comparisons EGFR, RAD21, OGG1, PARP2, SMARCAD1, BCCIP, TREX2, MCM7, UBE2A, INIP (SSBIP1), LIG1.Analysis revealed differentially expressed genes arranged in sets which are characteristic for more than two comparisons studied as well as unique ones for given comparisons . ThroughAP5S1, APBB1 (FE65), ASF1A, INTS3, JMY, NPAS2, PRRX1, RTEL1, SIRT7, SPIDR, TOP3A, USP51, VCP (p97), ZBTB1) were differentiating the AAA and control groups; twelve , HERC2, HSPA1A, NBN (NBS1), NFRKB, HMGN1, RAD17P1, USP1, TRRAP) were exclusively characteristic of the CVD and control group comparison; and eight (ACTR2 (ARP2), POLM, RECQL, SAMHD1, TERF2IP (RAP1), TOP1, UBE2V2 (MMS2), ZMPSTE24 (STE24)) had a dissimilar pattern of expression between the LEAD and CVD groups, while only four  distinguished LEAD from AAA patients. Their gene products are mainly related to responsibility for various DNA repair-related and genetic information maintenance processes, such as non-homologous end joining (NHEJ), double-strand breaks (DSBs) repair, transcription, DNA replication and chromosome maintenance . Overrepresentation analysis was performed to functionally annotate upregulated and downregulated gene sets selected from every comparison using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v2021 ,28 and t account .p = 7.9 × 10−10), “double-strand break repair” (p = 2.1 × 10−8), “regulation of response to DNA damage stimulus” (p = 3.3 × 10−8), “regulation of DNA repair” (p = 2.1 × 10−7) but also “chromosome organization” (p = 2.3 × 10−8), while upregulated genes were connected to “double-strand break repair” (p = 4.9 × 10−13), “regulation of double-strand break repair” (p = 7.9 × 10−7) and terms related to homologous recombination, such as “HDR through Homologous Recombination (HRR)” (p = 1.9 × 10−5) and the regulation of the DNA repair process, such as “regulation of DNA metabolic process” (p = 1 × 10−5) and “regulation of response to DNA damage stimulus” (p = 2.3 × 10−5). Downregulated genes in the CVD vs. control group were annotated to terms “double-strand break repair” (p = 3.4 × 10−3) and also terms connected to regulation, such as “regulation of DNA repair” (p = 3.4 × 10−3) and “regulation of DNA metabolic process” (p = 1.4 × 10−3), whereas upregulated genes represented terms like “double-strand break repair” (p = 1.7 × 10−5), “nucleotide excision repair” (p = 4.2 × 10−6) and various terms connected to “regulation of DNA repair” (p = 2.2 × 10−4). Downregulated genes in the LEAD vs. AAA group were primarily associated with “nucleoplasm” (p = 4.4 × 10−2), “double-strand break repair” (p = 0.0014) and “response to radiation” (p = 4.9 × 10−2), while upregulated ones, apart from terms connected to “double-strand break repair” (p = 1.9 × 10−6) and “regulation of double-strand break repair” (p = 0.001), were also associated with terms regarding “chromosome organization” (p = 3.2 × 10−5), “histone modification” (p = 6.2 × 10−4), and “chromatin remodeling” (p = 0.003). Upregulated genes in the LEAD vs. CVD group were ascribed to terms like “double-strand break repair” (p = 3.2 × 10−10), “DNA recombination” (p = 1.8 × 10−6) and “double-strand break repair via homologous recombination” (p = 1.5 × 10−4). Moreover, terms associated with various DNA repair regulatory processes, such as “regulation of DNA metabolic process” (p = 2.7 × 10−5) or “regulation of double-strand break repair” (p = 9.7 × 10−5) were present. Characteristic associations of downregulated genes from this comparison were, for example, “double-strand break repair via nonhomologous end joining” (p = 7.3 × 10−5) and “non-recombinational repair” (p = 2.55 × 10−4). Single genes from the LEAD vs. control and AAA vs. CVD comparisons were associated with terms like “histone mRNA catabolic process”, ATM; “Nucleotide Excision Repair”, “Base excision repair” and “Mismatch Repair”, LIG1; and “Nucleotide Excision Repair”, “HDR through Homologous Recombination (HRR) or Single Strand Annealing (SSA)”, “DNA Double-Strand Break Repair”, POLE4.The results showed that every comparison has been enriched with common terms like “DNA repair”, “DNA metabolic process”, “cellular response to DNA damage stimulus” and “cellular response to stress”, among others associated with nucleic acid metabolism. Differences in more specific terms were noticed between comparisons. Downregulated genes in the AAA vs. control group were primarily associated with “regulation of DNA metabolic process” (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) (accessed on 12 July 2022) [p below 0.05 were harvested  was performed for gene ontology categories in R environment using package clusterProfiler v 4.4.4. (ly 2022) ,30. In oarvested  and up tIn AAA vs. control, GO categories associated with transcription and regulation of transcription were downregulated 1.86-fold, whereas GO categories connected to “DNA–templated DNA replication” and “mitochondrion” were upregulated 1.64- and 1.68-fold, respectively. CVD vs. control categories describing nucleotide-excision repair were upregulated 1.77–1.98-fold, while those indicating various cell growth-related processes and their regulation were downregulated 1.91- to 1.98-fold. There were only upregulated GO categories in LEAD vs. AAA, including “regulation of double-strand break repair via homologous recombination” and “chromatin” with fold changes of 1.68 and 1.64, respectively. In LEAD vs. CVD, a cluster of more general categories emerged, such as “histone modification” (which increased 1.6-fold) or “proteolysis”; various references to catabolic processes and regulation were upregulated 1.81-fold.U test was used in the analysis of categorical variables (gender and smoking status). A statistical significance of p < 0.05, corrected by the Benjamini–Hochberg false discovery rate, was set as a selection criterion for both tests. Genes selected from the correlation analysis are presented in PRRX1, USP51, VCP were correlated, albeit moderately, with both age and BMI in the AAA vs. control group. Apart from that, single genes were also moderately correlated either with BMI or age in different comparisons. Therefore, the differential character of their expression patterns may be determined to some extent by differences in age and BMI between studied groups (APBB1 (p = 0.036) expression levels were correlated with smoking status and POLM (p = 0.015) and XRCC6 (p = 0.023) with gender  are all risk factors for cardiovascular diseases; thus, it is difficult to find a characteristics-matched group of control subjects. Due to differences in those traits between control and patient groups, statistical analysis regarding those traits was performed. Spearman rank correlation test was used in the analysis of continuous variables , while a two-sided Mann–Whitney d groups . There wh gender .DNA has the most important biological function; it bears the information of a blueprint of the living entity. Despite that, this crucial biological polymer is under constant threat of damage through the action of a plethora of chemical and physical factors.RAD21) [DEK) [MORF4L2 [FZR1 [SIRT7) [USP10) [TRRAP [NPAS2 [p < 0.05) and indicated by UVE-PLS to be informative were selected. Relationships between the expression of selected genes and age, BMI, gender and smoking habits were also assessed.It was earlier established that some DNA repair-related genes and their protein products are also engaged in various vascular-related processes, such as vascular endothelial growth factor A (VEGFA) function (RAD21) , muscle 1) [DEK) , atheros[MORF4L2 , FZR1 [3L2 [FZR1 ), neoint [SIRT7)  and foam [USP10) , and hav) [TRRAP , NPAS2 [P [NPAS2 ). This pAll additional comments and information regarding genes described in the main text, along with an introductory description of DNA damage metabolism pathways and extended discussion, were placed in Downregulated genes in the AAA vs. control group were primarily associated with various terms related to the regulation of DNA metabolism and repair, but also to transcription and the regulation of transcription, which may indicate decreased DNA repair activities associated with the transcription of genes required for DNA repair. On the other hand, terms describing the regulation of double-strand break repair, nucleotide-excision repair and various terms related to homologous recombination were overrepresented, leading to assumption that DSBs may be pronounced in AAA.TRRAP, PNKP, OGG1, NFRKB for “regulation of DNA repair” and AP5Z1, TRRAP, EME2, PNKP, OGG1, ARID2 for “double-strand break repair”) and downregulated  for each term, different in composition. This could be indicative of different modes of regulation of DNA repair present in CVD patients. Downregulated genes in the LEAD vs. AAA group were primarily associated with the replication of DNA, and “regulation of DNA metabolic process”. The term “double-strand break repair”, similarly to CVD, had ascribed both upregulated  and downregulated  groups of genes. This again points to DSBs and regulation of their repair as prominent features in those two diseases. Upregulation of terms connected to histone modification and chromatin may reveal chromatin remodeling processes distinguishing LEAD and AAA. While upregulated genes in LEAD vs. CVD were ascribed to terms like double-strand break repair and homologous recombination, there was significant downregulation of genes responsible for repair of DSBs via the NHEJ pathway, indicating that HR is the main pathway of DSBs’ repair in LEAD, in comparison with CVD.In CVD vs. control, genes ascribed to term “double-strand break repair” and “regulation of DNA repair” form two distinct groups, both upregulated  was performed for comparisons where more than ten genes were shown as differentiating, namely: AAA vs. control, CVD vs. control, Lead vs. AAA and LEAD vs. CVD. Results are shown in Due to their detrimental effects and toxicity, double-strand breaks require rapid detection, safeguarding and prompt repair. This forces the mutual action of many proteins. The repair process is not only directed at enzymatic activities processing damaged DNA; it is also highly regulated by accessory proteins ,40.PNKP, POLE, UPF1  [POLE) [UPF1) [PNKP, POLE and UPF1, REQL has been found as upregulated only in LEAD vs. CVD.Protein products of those genes contribute to the processing of free DNA ends (PNKP) , the sen) [POLE) ,43 and t) [UPF1) . REQL is) [UPF1) ,46. ContXRCC6 (Ku70), EGFR, INIP (SSBIP1) and INTS3. Proteins encoded by these genes play a critical role in efficient HR–dependent repair of DSBs and ATM-dependent signaling pathways [The second group of genes involved in DSB repair is composed of gene-encoding proteins engaged mainly in DNA binding, with less consistent expression changes through the compared groups. It includes pathways ,48.It is tempting to assume that revealed dependencies in expression changes could be indicative of diverse regulation of the same process in various diseases, creating the possibility of targeted therapies and diagnosis.PARP2, SMARCAD1, ARID2 (Baf200) (in a set of genes common for at least two comparisons) and AP5S1, APBB1 (FE65), ASF1A, SIRT7, SPIDR, USP51 and VCP (p97) (unique for the AAA vs. control group comparison) and SAMHD1 (exclusive to the LEAD vs. CVD comparison).The third group of genes consists of genes responsible for the regulation of the DSB repair process. It includes AP5S1, APBB1, ASF1A, SIRT7, SPIDR, USP51, VCP).The AAA vs. control group comparison was the richest in uniquely differentially expressed genes (eighteen positions). Among them, eight genes were ascribed to regulation of the DSBs repair process, on the basis of cited literature  and unique for only one comparison  contributing to homologous recombination [There were other genes present in sets common for at least two comparisons  transcription was examined [To further support engagement of homologous repair of DSB in the presented diseases, non-homologous end joining-related gene  gene, encodes protein taking part in NER and HR [Oxidative stress is both the crucial hallmark of cardiovascular diseases and their causative factor . The MRGR and HR ,72. DownCardiovascular diseases have long been described as ‘multifactorial’ , involviATM and UVSSA) to exclusiveness to only one comparison.Gene contribution revealed in our studies and according to the literature analysed varies from the regulation of a vascular-specific processes to commitment to a specific risk factor or cardiovascular system defects. The transcription pattern is also variable, ranging from four comparison occurrences  receptors , musclepression .NPAS2 (MOP4), PRRX1, RTEL1, SIRT7, USP10, VCP (p97). In the LEAD vs. CVD group, there were three such genes: ACTR2 (ARP2), SAMHD1, TERF2IP (RAP1), HERC2. In CVD patients (CVD vs. control group), there were two characteristic genes: HSPA1A, TRRAP; only one appeared in the LEAD vs. AAA comparison (CLK2).Gene sets correlated with both vascular-related processes/terms and DNA repair, unique for one comparison, only had the following composition. In AAA patients (AAA vs. control group) there were six genes with differential expression: NPAS2 [HERC2 [CLK2 [Interestingly, besides correlation with known vascular diseases and DNA repair, some genes point to the circadian oscillator being an important element of those conditions: NPAS2 , HERC2 [2 [HERC2  and CLK2C2 [CLK2 . This maC2 [CLK2 .NPAS2 (MOP4), PRRX1, RTEL1, SIRT7, USP10, VCP (p97)AAA vs. control group: Pathological vasculogenesis and arteriolar remodeling are hallmarks of different vascular diseases; both PPRX and RTEL1 are known to be engaged in those processes ,83, and SIRT7 gene product shapes VSMCs proliferation and migration in neointimal formation following vascular injury [The r injury , while Ur injury ,86. Morer injury ,88.ACTR2 (ARP2), SAMHD1, TERF2IP (RAP1)LEAD vs. CVD group: These genes are associated with various processes and correlated with various vasculopathies. They contribute to pathological cell motility, enhancing neointima formation after vascular injury (ACTR2 ), cerebrCVD vs. control group: HSPA1A (HSP70), TRRAPHSPA1A may be an element of compromised tolerance for stresses present in the course of CVD, compromising defense mechanisms including DNA repair [Decreased expression of A repair . The TRRA repair , and posA repair .ATM, POLE and POLE4, WHSC1 (NDS2), SMARCAD1, ARID2 (Baf200).DNA in eukaryotes tightly interacts with many specialized proteins, such as histones, building a highly organized entity called chromatin. Current NGS analysis did detect some of differentially expressed genes whose products are needed in various chromatin remodeling processes. In group of genes ascribed to more than one comparison, those were WHSC1; meanwhile, HMGN1 binds to 147-bp nucleosome core particle in purely structure-dependent manner [The ATM kinase is the node factor in a vast biochemical network of reciprocal interactions, signaling the presence of DNA damage and controlling the repair process . Recentlt manner  and facit manner ,100. Altt manner  ACTR2 [1t manner ,103 and t manner , has alsDifferential expression of genes related to chromatin remodeling clearly indicates the important contribution of this process in the regulation of DNA damage in the presented vascular diseases. What is interesting, and could be assumed, is that similarly to DNA DSBs repair, the regulation of various chromatin–related processes is an important factor to distinguish between vascular diseases. Those differences could have a tremendous impact on disease course and presentation.UBE2A, USP10, USP51, ZBTB1, HERC2, and SHPRH.One of potent mechanisms of protein function regulation is ubiquitination, defined as a covalent binding of ubiquitin protein to various other proteins. Current NGS analysis did detect some of differentially expressed genes, whose products are needed in various ubiquitin-related processes. Those genes were UBE2A (RAD6A) gene encodes an enzyme whose function is monoubiquitination of PCNA and taking part in DNA damage response to ionizing radiation [UBE2A was characteristic of LEAD in comparison with AAA and CVD.The adiation ,106. UprUSP10 and USP51 are deubiquitinases with increased expression in AAA patients. USP10 deubiquitinates and stabilizes MutS Homolog 2 (MSH2), which is the key DNA mismatch repair protein . USP51 iThe HERC2 protein is an E3 ubiquitin ligase negatively regulating both HR and NER ,109. UprPresented results show a connection between DNA damage responses and ubiquitin metabolism, showing possible different modes of regulation of DNA damage. Moreover, these seem to be specific for presented diseases, having potential value in both specific diagnosis and treatment.APBB1 expression levels were correlated with smoking status, and POLM and XRCC6 with gender , AAA (7 patients), CVD (7 patients) and control group of healthy volunteers (7 persons). All subjects were assessed by a vascular surgeon in Independent Public Clinical Hospital No. 1 in Lublin. All persons gave informed and signed consent. All mentioned procedures were performed in accordance with the Declaration of Helsinki. The Ethics Committee of Medical University of Lublin approved the study design (resolution No. KE–0254/341/2015). The clinical characteristics regarding all participants are presented in Next-generation sequencing was the method of choice for the assessment of transcriptomic profiles of PBMCs from study participants. An entire detailed methodology with accompanying data regarding sequencing data quality was provided in previous papers ,23,24.Briefly, blood samples were subjected to PBMCs isolation using density gradient centrifugation with Gradisol L reagent . Concisely, total RNA specimens  were isolated from PBMCs samples of vascular diseases patients and randomly selected controls using TRI Reagent Solution . The quality of total RNA samples was assessed using the Agilent 2100 Bioanalyzer instrument with the Agilent RNA 6000 Pico Kit for total RNA samples and the Agilent DNA 1000 Kit for libraries  and supp. Subsequhttps://www.r-project.org) (accessed on 18 May 2021) and appropriate packages according to the provided reference manuals were used for statistical analyses and data visualizations. Kruskal–Wallis rank sum test  was applied to assess the statistical significance of differences in age and BMI between the LEAD, AAA, CVD and control groups. A two-sided Fisher’s exact test (fisher.test function in R) was applied to assess the statistical significance of differences in gender and smoking status between the studied groups.R environment  ,112 methhttps://bioconductor.org/packages/release/bioc/html/DESeq2.html) (accessed on 18 May 2021) on expression data devoid of genes with mean number of reads lower than one. Differentially expressed genes were considered as statistically significant when the criterion of a p value below 0.05 after Benjamini–Hochberg false discovery rate correction was met.DESeq2 analysis was carried out using a DESeq2 1.26.0 package  (https:/https://cran.r-project.org/web/packages/plsVarSel/index.html) (accessed on 18 May 2021) was used to perform UVE-PLS analysis. The initial data was transformed by regularized log normalization (rlog function in the DESeq2 package). 1000 iterations, a 0.75 ratio for splitting into trained and tested data subsets and a minimum reliable score equal to 5 cutoff threshold were the values used in UVE-PLS analysis. The number of PLS  components for UVE-PLS analysis  (accessed on 19 May 2021) was used to conduct the ROC analysis.The pROC package 1.16.2  (https:/https://ggplot2.tidyverse.org) (accessed on 29 August 2021).Boxplots were generated using the ggplot2 3.3.0 package  was performed using a Spearman rank correlation test implemented in the Hmisc package 4.4–0. (https://cran.r-project.org/web/packages/Hmisc/index.html) (accessed on 29 August 2021).The statistical significance of categorical characteristics (gender and smoking habits) was assessed with a two-sided Mann–Whitney https://david.ncifcrf.gov/home.jsp (accessed on 11 July 2022)) [Overrepresentation functional analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) (y 2022)) ,28. The https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) (accessed on 12 July 2022) [Gene set enrichment analysis (GSEA) was performed in R environment with package clusterProfiler v4.4.4. (ly 2022) ,30. AnalBlood mononuclear cells were widely used in the assessment of not only various atherosclerotic risk factors like hypertension , type 2 The presented research sheds light on DNA damage repair in patients with three vascular diseases: AAA, CVD and LEAD. In-depth data analysis revealed gene sets that could be ascribed to numerous DNA repair processes, pointing especially to the prominent homologous repair presence in all probed diseases. Data also indicate a significant crosstalk between diverse pathways interrelated with DNA repair, encompassing chromatin remodeling, cohesin metabolism and function and ubiquitin turnover, and oxidative stress presence and management. These characteristics show significant commonalities in processes contributing to the presented vascular diseases. Striking differences appear when the analysis takes into account the regulatory component present in the examined diseases. Data suggest that gene-encoding proteins responsible for control and fine–tuning of mentioned processes exhibit the most interesting dissimilarities in expression. This could be the key that describes and distinguishes one condition from the other, and may explain the relative richness of their clinical presentations and courses. This is also a prerequisite for the creation of new diagnostic methods and therapies utilizing DNA repair as the target of intervention.


Potentilla species, namely P. alba (PAL7), P. argentea (PAR7), P. grandiflora (PGR7), P. norvegica (PN7), P. recta (PRE7), and the closely related Drymocalis rupestris (syn. P. rupestris) (PRU7), were analysed for their cytotoxicity and antiproliferative activities against human colon adenocarcinoma cell line LS180 and human colon epithelial cell line CCD841 CoN. Moreover, quantitative assessments of the total polyphenolic (TPC), total tannin (TTC), total proanthocyanidins (TPrC), total flavonoid (TFC), and total phenolic acid (TPAC) were conducted. The analysis of secondary metabolite composition was carried out by LC-PDA-HRMS. The highest TPC and TTC were found in PAR7 /g extract, respectively) and PN7 . The highest TPrC, TFC, and TPAC levels were found for PAL7 /g extract, 71.85 mg rutin equivalents (RE)/g extract, and 124.18 mg caffeic acid equivalents (CAE)/g extract, respectively). LC-PDA-HRMS analysis revealed the presence of 83 compounds, including brevifolincarboxylic acid, ellagic acid, pedunculagin, agrimoniin, chlorogenic acid, astragalin, and tiliroside. Moreover, the presence of tri-coumaroyl spermidine was demonstrated for the first time in the genus Potentilla. Results of the MTT assay revealed that all tested extracts decreased the viability of both cell lines; however, a markedly stronger effect was observed in the colon cancer cells. The highest selectivity was demonstrated by PAR7, which effectively inhibited the metabolic activity of LS180 cells (IC50 = 38 μg/ml), while at the same time causing the lowest unwanted effects in CCD841 CoN cells . BrdU assay revealed a significant decrease in DNA synthesis in both examined cell lines in response to all investigated extracts. It should be emphasized that the tested extracts had a stronger effect on colon cancer cells than normal colon cells, and the most significant antiproliferative properties were observed in the case of PAR7 (IC50 LS180 = 174 μg/ml) and PN7 (IC50 LS180 = 169 μg/ml). The results of LDH assay revealed that all tested extracts were not cytotoxic against normal colon epithelial cells, whereas in the cancer cells, all compounds significantly damaged cell membranes, and the observed effect was dose-dependent. The highest cytotoxicity was observed in LS180 cells in response to PAR7, which, in concentrations ranging from 25 to 250 μg/ml, increased LDH release by 110%–1,062%, respectively. Performed studies have revealed that all Potentilla species may be useful sources for anti-colorectal cancer agents; however, additional research is required to prove this definitively.Cinquefoils have been widely used in local folk medicine in Europe and Asia to manage various gastrointestinal inflammations and/or infections, certain forms of cancer, thyroid gland disorders, and wound healing. In the present paper, acetone extracts from aerial parts of selected  Potentilla species, known as cinquefoils, are widely used, since they are well known in traditional medicine throughout the Asian and European continents as valuable phytomedicines in a remedy inter alia against diarrhoea, ulcers, fever, jaundice, oral inflammations, topical infections, and thyroid gland disorders , Hep G2 (human hepatocellular carcinoma) and T84 (human colonic adenocarcinoma), while extracts and fractions from aerial parts of P. alba decreased proliferation and viability of HT-29 (human colon adenocarcinoma)  . Howeverisorders . Moreove cancers . A numbeoperties . Moreovercinoma) .Potentilla species, similarly to other species from this genus, would exhibit broad pharmacological potential. Therefore, the primary aim of our study was to assess their cytotoxicity and antiproliferative activities against human colon adenocarcinoma cell line LS180 and human colon epithelial cell line CCD841 CoN. Moreover, we identified the marker metabolites present in extracts through LC-PDA-HRMS analysis to uncover correlations between the qualitative chemical composition of extracts and their possible mechanism of action.We hypothesized that aerial parts of selected O-glucuronide, kaempferol 3-O-glucuronide and isorhamnetin 3-O-glucoside were obtained from Extrasynthese  (+)-Catechin, (-)-epicatechin and gallic acid were the products of Carl Roth . Quercetin 3-O-glucoside, quercetin 3-O-rutinoside, kaempferol 3-O-glucoside (purity >96%) were isolated from flowers of Ficaria verna L. Hud (Ranunculaceae)  was isolated from aerial parts of Rubus saxatilis L. (Rosaceae) (Rubus caesius L. (Rosaceae)  were isolated from aerial parts of Drymocalis rupestris (L.) Soják (Rosaceae) (O-methyl ether 4-O-xyloside (purity >96%) were isolated from aerial parts of P. recta (O-caffeoylquinic acid (purity >96%) were isolated from leaves and inflorescences of Arctium tomentosum Mill. (Asteraceae) . Quercetin 3-ulaceae) . Quercetosaceae)  and peduosaceae) . Quercetosaceae) . AgrimonP. recta . Apigenieraceae) . All othP. alba (ind. sem. 354), P. grandiflora (ind. sem. 758), P. norvegica (ind. sem. 303), P. recta (ind. sem. 1549) and P. rupestris (ind. sem. 763) were kindly provided by the Botanical Garden of Vilnius University , Giardino Botanico Alpino , Hortus Botanicus Universitatis Masarykianae  and Hortus Botanicus University of Tartu . Plants were cultivated in common plots at the Medicinal Plant Garden at the Medical University of Białystok , and aboveground materials were collected in June-August 2016–2019. Aerial parts of P. argentea were collected in June-July 2017–2019 from natural habitat, at Puszcza Knyszyńska . The taxonomic identification of plant material was carefully authenticated by one of the authors (M.T.). Voucher specimens of P. alba (PAL-17039), P. argentea (PAR-02009), P. grandiflora (PGR-06020), P. norvegica (PNO-08024), P. recta (PRE-06019) and P. rupestris (PRU-06021) have been deposited at the Herbarium of the Department of Pharmacognosy, Medical University of Białystok (Poland). Collected dried materials were subsequently finely grounded with an electric grinder and stored in air-tight containers at ambient temperature. Powdered dry plant materials (2.0 g each time) were separately submitted to ultrasound-assisted extraction with 70% acetone (3 × 50 ml) using an ultrasonic bath  at a controlled temperature (40 ± 2 °C) for 45 min in a 1:75 (w:v) solvent ratio. The obtained raw extracts after solvent evaporation were diluted with water (50 ml) and subsequently portioned with chloroform (10 × 20 ml). The acetone extracts were obtained using this method for P. alba (PAL7), P. argentea (PAR7), P. grandiflora (PGR7), P. norvegica (PN7), P. recta (PRE7) and P. rupestris (PRU7).Seeds of five species, namely v/v) and the mixture was allowed to react for 3 min. Thereafter, 75 µl of 1% Na2CO3 solution was added and the prepared mixture was incubated for 2 h at ambient temperature. The absorbance was measured at 760 nm using a microplate reader EPOCH2 BioTech . The TPC determination was repeated at least three times for each sample solution. Obtained results were expressed as milligrams of gallic acid equivalents per Gram of dry extract (mg GAE/g extract).The total phenolic content (TPC) was measured by the Folin-Ciocalteu assay with some modifications . Brieflyv/v). After 3 min 75 µl of 1% Na2CO3 was added and the mixture was allowed to stand for 2 h at room temperature. Thereafter the absorbance of each sample (A1) was recorded at 760 nm using a EPOCH2 microplate reader. Subsequently, the second part of aliquots of 0.5 ml each were mixed with 10 mg of hide powder. These preparations were shaken for 1 h without light and then centrifugated. A 25 µl of supernatants were assayed for total polyphenolics as described above and the absorbance of each sample (A2) was recorded at 760 nm. Afterwards, the total tannin content was determined by subtraction of absorbances of total polyphenols (A1) from total non-tannin polyphenols (A2) and the obtained absorbance values were referred to a gallic acid calibration curve to obtain their values as milligrams of gallic acid equivalents per Gram of dry extract (mg GAE/g extract). The determination of TTC was repeated at least three times for each sample solution.The total tannin content (TTC) of each extract was measured by the employment of the protein-binding method and Folin-Ciocalteu assay described in the European Pharmacopoeia 10th ed . with moThe total proanthocyanidin content (TPrC) was analysed with the employment of a 4-dimethylamino-cinnamaldehyde (DMCA) reagent . The ana3) colorimetric method (w:v) AlCl3 solution. The mixture was kept at ambient temperature for 10 min. Then the absorbance of the mixture was recorded at 415 nm using a EPOCH2 microplate reader. The TFC determination was repeated at least three times for each sample solution. TFC was expressed as milligrams of rutin equivalents per Gram of dry extract (mg RE/g extract).The total flavonoid content (TFC) of each extract was determined using the previously described aluminium chloride  determination was carried out using the procedure with the use of Arnov’s reagent (1 g of sodium molybdate and 1 g of sodium nitrate dissolved in 10 ml of distilled water) . A 30 µl/z range. The separation was performed using a Kinetex XB-C18 column . The mobile phases were ultra pure water (A) and acetonitrile (B) with 0.2% formic acid. The separation was achieved by a gradient of 0–3 min 65% B; 3–35 min 1% B, 35–80 min 12% B, 80–113 min 45% B, extended by 7 min of equilibrating time. The flow rate was 0.2 ml/min, and the column temperature was maintained at 35 ± 0.8°C. The UV-vis spectra were recorded in the range of 190–540 nm with selective wavelength monitoring at 280 and 360 nm. Data were processed with the employment of MassHunter Qualitative 10.0. Analysis software. Compounds were characterized based on UV–Vis and MS spectra and retention time of standards.Evaluation of the secondary metabolite composition of each extract was conducted using an Agilent 1260 Infinity LC chromatography system coupled to a photo-diode array (PDA) detector and 6230 time-of-flight (TOF) mass spectrometer . The MS conditions were as follows: electrospray ionization (ESI) source in both negative and positive ionization mode, drying and sheath gas flow 11 L/min and temperature of 350°C, nebulizer pressure of 60 psi, capillary voltages of 2,500 and 4500 V for negative and positive ion modes, respectively and fragmentor experiments at 60, 180 and 320 V. The data were collected in the 120–3,000 mHuman colonic epithelial cell line CCD841 CoN was purchased from the American Type Culture Collection . Human colon adenocarcinoma cell line LS180 was obtained from the European Collection of Cell Cultures . Cell cultures were conducted in accordance with the guidelines of the collections in which they were purchased.4 cells/mL. The following day, the culture medium was exchanged for fresh medium supplemented with investigated extracts or 25 μM 5-fluorouracil (5-FU). After 48 h of cell treatment, the compounds’ antiproliferative effect was determined using Cell Proliferation ELISA BrdU, following the manufacturer’s instructions , while the compounds’ cytotoxicity was examined by the In Vitro Toxicology Assay Kit Lactate Dehydrogenase Based according to the manufacturer’s instructions . Furthermore, cell viability in response to 48 h of exposure to investigated compounds was determined by MTT assays. A detailed description of the execution of the above-mentioned assays was presented by Langner and co-authors  was calculated using GraphPad PRISM.The analysed data were presented as the mean ± SEM. Statistical analyses were performed using One way-ANOVA with the Tukey PAR7 and PN7, followed by PRU7, were found to contain the highest TPC  and TTC . PAL7 had the lowest TPC and TTC values (159.87 and 84.89 mg GAE/g). However, PAL7 was found to contain the highest TPrC (21.28 mg CE/g extract), while the other extracts had low proanthocyanidin content. The TFC levels for all tested samples were found to be similar, with the highest values for PAL7 and PAR7 . Moreover, the highest TPAC values were revealed for PAL7 and PN7 .In the first set of experiments, the studied extracts were examined for their TPC, TTC, TPrC, TFC, and TPAC using colorimetric methods. The obtained results are presented in Potentilla species were analysed via LC-PDA-HRMS. The analysis demonstrated the presence of 83 compounds, predominately polyphenolic compounds, ascribed to hydrolysable and condensed tannins, flavonoids, and phenolic acids. Hydrolysable tannins were present in all extracts with the exception of PAL7, primarily represented by ellagitannins, such as pedunculagin α and β , agrimoniin (69), laevigatin isomers , galloyl-HHDP-glucose isomers , galloyl-bis-HHDP-glucose isomers , ellagic acid (51), ellagic acid 3,3′-di-O-methyl ether 4-O-xyloside (75), ellagic acid 3′-O-methyl ether 4-O-xyloside (54), and ellagic acid 3′-O-methyl ether 4-O-arabinoside (70). PRU7 showed the presence of gallotannins, such as tri-, tetra-, and pentagalloylglucose isomers . Moreover, the degradation products of hydrolysable tannins, namely brevifolincarboxylic acid 19) and brevifolin (33), were found. On the other hand, PAL7 and PAR7 were rich in the condensed tannins, such as (+)-catechin 13) and (-)-epicatechin (27), and oligomeric procyanidins, such as procyanidin B1 (12), B2 (20), and C1 (37). A number of flavonoids were detected and characterized, including apigenin 78) and its O-hexoside (68), isorhamnetin , kaempferol , and quercetin  derivatives. In addition, phenolic acids were also present in extracts, such as gallic acid (1), caffeic acid derivatives , p-coumaroylquinic acid isomers , and N1, N5, N10-tricoumaroyl spermidine (83). The detailed chromatographic data are shown in To unveil the secondary metabolite composition, acetone extracts of selected Potentilla extracts. Cells were exposed to either culture medium (control) or extracts (25–250 μg/ml) for 48 h and, afterward, an MTT test was performed. As presented in PN7, which at the highest tested concentration decreased LS180 cell proliferation by 87.3% (IC50 PN7 LS180 = 32 μg/ml), while the weakest effect was noted for PAL7, which at a concentration of 250 μg/ml inhibited cancer cells division by 57.5% (IC50 PAL7 LS180 = 182 μg/ml). In the case of colon epithelial cells, the strongest reduction (49.5%) of their metabolic activity was observed after exposure to 250 μg/ml PAL7 (IC50 PAL7 CCD841 CoN = 233 μg/ml), while the weakest effect, as reflected by the IC50 value, was for PAR7 . Although all investigated extracts affected both normal and cancer colon cells, LS180 cells were more sensitive to the tested compounds. Comparing the metabolic activity in both analysed cell lines in response to extracts at the corresponding concentrations, greater sensitivity of cancer cells was observed in the entire range of analysed concentrations in the case of PAR7, PRE7, PRU7, and PN7, while PGR7 showed statistically significant differences in concentrations from 50 μg/ml to 250 μg/ml. Even PAL7 at the highest tested concentrations (150 and 250 μg/ml) strongly inhibited the viability of cancer cells than colon epithelial cells. As a positive control of the experiment, 5-fluorouracil (5-FU) at a concentration of 25 µM was used ; PRE7 ; and both PAR7 and PRU7 (250 μg/ml). In the case of colon cancer cells, PGR7, PAR7, PRE7, PRU7, and PN7 at concentrations from 50 to 250 μg/ml and PAL7 at a concentration of 250 μg/ml showed a stronger anti-metabolic effect than the analysed cytostatic.In the next set of experiments, the viability of both human colon epithelial cell line CCD841 CoN and human colon adenocarcinoma cell line LS180 was examined in response to the investigated was used . The metPotentilla extracts was assessed in the abovementioned cell lines using BrdU assay  and the greatest decrease of DNA synthesis in LS180 cells in response to the extracts at a concentration of 250 μg/ml (cell proliferation was reduced by 63.1% (PAR7) and 71.1% (PN7)). Colon cancer cell division was least inhibited by PAL7 (IC50 PAL7 LS180 = 12 mg/ml), which at the highest tested concentration decreased DNA synthesis by only 9.1%. The investigated extracts also affected the proliferation of colon epithelial cells and statistically significant inhibition of DNA synthesis was noted in response to all compounds at concentrations of 150 and 250 μg/ml, while in the case of PN7, PAR7, PRU7, and PGR7 the antiproliferative effect was observed also at a concentration of 100 μg/ml. Similar to the colon cancer cells, epithelial cells were the most sensitive to PN7 and PAR7, which at a concentration of 250 μg/ml reduced their proliferation by 58.4% and 53.4%, respectively (IC50 PN7 CCD841 CoN = 217 μg/ml and IC50 PAR7 CCD841 CoN = 217 μg/ml). The weakest antiproliferative effect in CCD841 CoN cells was observed after exposure to PAL7, which at the highest tested concentration inhibited cell division by only 6.7%. Studies have revealed the antiproliferative abilities of Potentilla extracts in both normal and cancer colon cells, nevertheless PGR7 at the highest tested concentration, as well as PAR7, PRU7, and PN7 at concentrations 150 and 250 μg/ml, inhibited DNA synthesis significantly more strongly in LS180 cells than CCD841 CoN cells. As presented in PAR7 and PN7 ; PRE7 ; and both PGR7 and PRU7 (250 μg/ml). The obtained data indicated a higher selectivity of the analysed cytostatic compared with examined extracts in the case of influence on DNA synthesis.In the next step, the antiproliferative activity of dU assay ; Table 3in vitro studies, extracts cytotoxicity was examined in CCD841 CoN cells and LS180 cells using LDH-based assay. As presented in PAR7, which in concentrations ranging from 25 to 250 μg/ml increased the LDH level by 110% and 1,062%, respectively. Very similar results were obtained after LS180 cell exposure to PRU7, which in the mentioned range of concentrations increased LDH release by 161% (25 μg/ml) and 956% (250 μg/ml). The weakest cytotoxic effect was noted in colon cancer cells treated with PAL7, which at the highest tested concentration caused an increase in the LDH level of 68%. Used as a positive control, 5-FU at a concentration of 25 µM was not cytotoxic against colon epithelial or colon cancer cells  increased the LDH level to 261, 210, 185, and 156%, respectively.In the last step of the er cells . The LDHPotentilla species are a source of a wide spectrum of secondary metabolites, mainly polyphenols, such as hydrolysable and condensed tannins, flavonoids and their glycosides, and phenolic acids, which show a variety of biological activities , proliferation (BrdU assay), and cytotoxicity (LDH assay). All investigated extracts decreased viability of both normal and cancer colon cells in a dose-dependent manner; however, LS180 cells were more sensitive to the tested compounds. The results of MTT assay indicated that the tested extracts effectively decreased the mitochondrial metabolism of human colon cancer cells, which could be associated with the presence of hydrolysable tannins in all extracts except the PAL7, which revealed the weakest anticancer effect. Moreover, the highest impact in decrease of cancer cells viability by PAR7, PN7 and PRU7 correlate with their highest TPC and TTC values. Agrimoniin was shown to have prominent antioxidative, anti-inflammatory, and anticancer effects. P. erecta directly inhibit UVB-induced cyclooxygenase-2 (COX-2) expression and production of PGE2 in human keratinocytes (HaCaT), as well as in an in vivo model, and inhibit epidermal growth factor receptor (EGFR) phosphorylation. Fragaria x ananasa, Rosaceae) containing 16.2% agrimoniin downregulated the mRNA expression of COX-2, IL-1β, IL-6, TNF-α, and iNOS in AOM/DSS-induced colon cancer in mice. BrdU assay revealed a significant decrease of DNA synthesis in both colon cancer and non-cancer cells in response to all investigated extracts. The strongest antiproliferation effect in cancer cells was observed after treatment with PAR7 and PN7. Those extracts revealed to posess the highest total polyphenol and tannin contents. Notably, the antiproliferative effect of 5-FU observed in colon cancer cells was significantly stronger than that of the examined extracts. Similarly, data collected from colon epithelial cells revealed that five out of six investigated extracts in higher concentrations inhibited DNA synthesis stronger than the positive control. The in vivo bioavailability of high weight ellagitannins is relatively low. Ellagitannins at neutral or alkaline pH are hydrolysed with the release of free ellagic acid, which exerts a number of biological activities , protein p53, and Bax, and decreasing expression of anti-apoptotic proteins, cleaved caspase 3, and Bcl-2. Moreover, the investigated compound causes G0/G1 arrest, inhibits the expression of metalloproteinases MMP-2 and MMP-9, and decreases the activity of the NF-κB signalling pathway  found that hydrolysable tannins selectively block calcium-activated chloride channels and form selective pores in the cell membrane  showed great chemopreventive potential, manifested by the effective elimination of colon cancer cells, causing both damage to their cell membranes and inhibition of their proliferation and metabolic activity, with a simultaneous lack of a cytotoxic effect on normal colon epithelial cells and a significantly weaker effect on their metabolism and DNA synthesis compared to cancer cells. While it is impossible to specify the extract with the greatest therapeutic potential, these studies unequivocally showed that PAL7 had the lowest anti-cancer potential in a cellular model of colon cancer.In conclusion, this study reports, for the first time, analysis of the LC-PDA-HRMS profile of acetone extracts of selected


Skin irritation test with reconstituted epidermis (Skin Ethic RHETM) showed that the formulation is non-irritating. Tyrosinase inhibition was maintained after skin permeation in vitro, in which microemulsion showed twice the inhibition of the conventional emulsion . The depigmenting effect of the microemulsion was finally confirmed in a 3D culture model of pigmented skin, in which histological analysis showed a more pronounced effect than a commercial depigmenting formulation. In conclusion, the developed microemulsion is a promising safe formulation for the administration of cutite fruit extract, which showed remarkable depigmenting potential compared to a commercial formulation.Here, we verify the depigmenting action of Pouteria macrophylla fruit extract (EXT), incorporate it into a safe topical microemulsion and assess its effectiveness in a 3D pigmented skin model. Melanocytes-B16F10- were used to assess the EXT effects on cell viability, melanin synthesis, and melanin synthesis-related gene transcription factor expression, which demonstrated a 32% and 50% reduction of intra and extracellular melanin content, respectively. The developed microemulsion was composed of Cremophor EL Pouteria macrophylla (Lam.) Eyma belongs to the Sapotaceae family and is found in the Brazilian Amazon region and other South American countries, including Bolivia, Peru, French Guiana, Suriname, Colombia, and Venezuela. Its fruits possess common names such as cutite, jarana, abiurana-cutite, taturubá, and abiu-cutite, among others. The fruit pulp can be consumed fresh or in the manufacture of ice cream and sweets, while the fruit peel is popularly used to treat dysentery -ethanesulfonic acid), SpanPouteria macrophylla (Lam.) Eyma existing in the Herbarium of the Museum Emílio Goeldi (MG239766), city of Belém, state of Pará, Brazil. Fruits were freeze-dried for 48 h and then extracted by percolation with ethanol assisted by ultrasound for 10 min, as previously described [The fruits were collected, and the aerial parts and fruits were identified by comparison with authentic vouchers of Goeldi MG39766, ciThe determination of GA, the biomarker of the cutite fruit ethanolic extract, was performed by liquid chromatography–mass spectrometry (LC-MS) using an equipment model 2020 from Shimadzu , connected to a Genius NM32LA nitrogen gas generator model  and mechanical vacuum pump , with dual-spray ionization source, acting on negative ionization mode by electrospray (ESI-), coupled with liquid chromatography system.A C18 reverse-phase column  was used as the stationary phase. The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) applied by gradient with a flow rate of 0.4 mL/min according to the schedule: A/B = 90/10 (0.01 min–1.5 min), 80/20 (1.6 min–3.5 min), 70/30 (3.6 min–5.5 min), 60/40 (5.6 min–7.5 min), 10/90 (7.6 min–10 min), and 90/10 (10.1 min–15 min). The oven temperature was maintained at 35 °C, and the injection volume of each sample was 2 μL. The limits of detection and quantification were 0.1 μg/mL and 0.3 μg/mL.v/v) FBS and 1% (v/v) of penicillin/streptomycin solution (1000 U/mL).The analysis of the cellular toxicity, depigmentation effect, and suppression of genes involved in melanogenesis by GA and EXT was performed in immortalized melanocyte cultures from murine melanoma (B16F10). The cells were cultured using DMEM supplemented with 10%  using DMSO as a solvent and GA was used at 10, 25, 50, 100, 200, and 400 μM. Cells incubated with vehicle only were considered as untreated controls. After 48 h, cells were washed with PBS, fresh DMEM was supplemented with FBS, and 5 mg/mL MTT was added, followed by plate incubation for 4 h in the dark. Once the MTT solution was carefully removed, DMSO was added to dissolve the formed formazan crystals. A spectrophotometer of microplates  was used to read the microplate at 570 nm. After deducting the optical density obtained from the blank (DMSO), the analysis was carried out. Data were normalized to the untreated controls, considered 100% viability, representing the mean ± SD of three independent experiments.EXT and standard analytical concentrations were tested to select the highest non-toxic concentrations for further experiments. B16F10 cells were seeded in 96-well plates at a density of 1 × 105 cells were plated in 6-well plates and incubated with 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) to induce melanogenesis and treated with GA or EXT, for 48 h. Extracellular melanin content was evaluated by collecting 100 μL of cell culture supernatant from each well. The reading was performed in a microplate reader  at 405 nm, and the analysis was performed after deducting the optical density obtained in the blank (DMEM). The intracellular melanin content was evaluated from cells harvested, lysed, and mixed with 200 μL of NaOH (1 M) for 16 h at room temperature (20 –25 °C). The absorbance was measured at 405 nm, and the analysis was performed after deducting the optical density obtained in the blank (NaOH). The melanin content of IBMX-stimulated cells was considered 100% and used to normalize melanin production in the experimental groups. Untreated samples received vehicle only and were used as the negative control.Melanin production was assessed regarding intracellular melanin production and secretion in the cell culture supernatant, as previously described by our group . BrieflyThe isolation of total RNA from the cells treated with IBMX, or IBMX and GA or EXT for 48 h was performed using the reagent trizol and following the manufacturer’s instructions to analyze MITF and TYR mRNA expression. First, the RNA concentration was determined by reading the absorbance at 260/280 nm on Nanodrop equipment . Next, the RNA samples were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit, following the manufacturer’s instructions. Finally, the mRNA levels were determined by qPCR using the SYBRTM Green Master Mix  and the following primers: GAPDH F - ACATCGCTCAGACACCATG, GAPDH R - TGTAGTTGAGGTCAATGAAGGG; MITF F: AGGACCTTGAAAACCGACAG, MITF R - GTGGATGGGATAAGGGAAAG; TYR F: AGCCTGTGCCTCCTCTAA, TYR R: AGGAACCTCTGCCTGAAA. Amplifications were performed by StepOne Plus equipment , and the data were analyzed by StepOne Software v2.3  using th® and Span® 80 were mixed in a ratio of 4:1 (w/w) under vigorous stirring. The surfactants were then dissolved in oil phase (ethyl oleate) in progressive ratios of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1 (w/w) at room temperature. The systems obtained from each mixture rested for 5 min and were then classified according to their physical aspect as “phase separation”, “milky”, “cloudy”, “flocculation”, or “possible microemulsion”.The microemulsion was obtained from a pseudoternary phase diagram as previously described . BrieflyAmong the region that forms microemulsion, the composition with the highest viscosity, a higher proportion of pH 4.5 HEPES buffer, and a lower concentration of surfactants was selected to continue the study. Subsequently, 37.3 mg/g of EXT, corresponding to 0.05% of GA content, was added to the chosen microemulsion and mixed with a glass stick for incorporation. The EXT was added at temperatures lower than 40 °C.The microemulsion was characterized by means of droplet size, polydispersity index (PDI), and zeta potential using a “Zetasizer Nano ZS” equipment  and pH using DM-22 equipment . Morphological analysis was also performed using diluted samples (1:1000) analyzed by transmission electron microscopy . The images were captured with a GATAN BioScan camera  using the Digital Micrograph 3.6.5 software .® AVC, 0.1% EDTA-disodium, 5% propylene glycol humectant, and 94.5% pH 4.5 HEPES buffer. Phase 2 was composed by 14% Polawax®, 1% liquid vaseline, 2% DC 556, 1% DC 2501, 0.1% BHT and 0.5% methylparaben and propylparaben. After mild heating (50 °C), phase 1 was poured into phase 2 under vigorous stirring to form the emulsion. After reaching room temperature, the emulsion was homogenized and centrifuged at 4000 rpm for 10 min to assure there would be no phase separation. The pH of the formulation was 4.0 ± 0.3. For comparison purposes, the same amount of EXT was incorporated by geometric dilution technique into the conventional emulsion (37.3 mg/g).An emulsion gel was obtained to serve as a conventional formulation control by preparation of two heated phases separately. Phase 1 comprised 0.4% Aristoflexn = 3 for each formulation). At 0, 1, 7, 15, and 30 days, samples were analyzed for droplet size, PDI, and zeta potential for the microemulsion only, and pH and GA content for both the microemulsion and the conventional emulsion formulations.Stability studies were carried out for 30 days with formulation samples stored in hermetically sealed Eppendorf at room temperature (20–25 °C) (w/v) was used as a positive control, and saline solution at 0.9% (w/v) as a negative control. The samples remained in contact with the RHE for 42 min in an oven at 5% CO2, 37 °C, and 95% humidity. After this period, each well was washed with 25 jets of 1 mL of saline solution, and the plate was stored for 42 h under the same conditions. Finally, the tissues were transferred to another 24-well plate containing 300 μL/well MTT bromide solution at 1 mg/mL (1:5 dilution at the time of use) and incubated for 2 h protected from light in an oven at 5% CO2, 37 °C, and 95% humidity. Subsequently, 750 μL of isopropanol was added under and 750 μL over the tissues for extraction. Then, the reading was performed by colorimetry in a microplate reader  at 570 nm. The study was carried out in triplicate, and the result was based on the optical density obtained from each sample minus the optical density of the blank (isopropanol).The irritative potential of the microemulsion and the conventional emulsion was evaluated using reconstructed human skin  provided by EpiSkin™ according to OECD guidelines described in test n. 439 . For the2) mounted with hydrophilic cellulose membranes  separating donor and receptor compartments [The release of GA from the EXT incorporated into the formulations was determined in vitro throughout 12 h using modified Franz-type diffusion cells (diffusion area = 1.3 cmartments . The don2) for 6 h. The donor compartments were filled with 500 mg of microemulsion or conventional emulsion. The system temperature was kept at 30 °C by a water bath. Afterward, the buffer samples were collected and the tyrosinase bioassay method described in In vitro cutaneous permeation studies were performed trough porcine skin using modified Franz-type diffusion cells  and fibroblasts . After polymerization, 2.5 × 105 human keratinocytes and 1.7 × 105 human melanocytes were seeded in RAFT: KGM-Gold Bullet Kit medium (1:1) on top of each lattice, and the skins were kept submerged in the culture medium for 24 h. The construct was transferred and maintained at an air-liquid interface for 10 days in a 5% CO2 incubator to allow complete keratinocytes stratification and differentiation. The medium was supplemented with tyrosine (0.25 mM) and NH4Cl (5 mM) and was replaced every 2 days to induce pigmentation. The skins were treated with 10 μL of the commercial product (kojic acid serum) or 10 μL of the microemulsion containing the EXT for 12 h. After this, skins were fixed in 10% buffered formalin at 4 °C for 12 h, followed by dehydration in solutions containing increasing concentrations of alcohol and xylene for paraffin inclusion. Paraffin sections (5 μm) were stained with Fontana-Masson. All images were obtained by optical microscopy (Nikon Eclipse- 20× and 40×). The experiment was performed in three independent replicates.The 3D pigmented reconstructed human skin model was first obtained by our group as previously reported. Basically, normal human melanocytes, keratinocytes, and fibroblasts were cultivated in specific growth medium for each cell type and maintained in an incubator at 37 °C containing 7.5% COp < 0.05, p < 0.01, p < 0.001, and p < 0.0001. Differences between datasets were verified by normality tests via one-way analysis of variance, followed by Tukey’s multiple comparison test.The pseudoternary phase diagram was constructed with Origin 8 . Statistical analyses were conducted using Prism 6 , in which the level of significance was set at The present study demonstrated the depigmenting potential of cutite fruit extract for the first time. Melanocyte cultures showed decreased intracellular melanin production and secretion, along with suppression of melanogenic related genes MITF and TYR. Furthermore, when incorporated in a microemulsion formulation, the extract was slowly released and provided a targeted delivery to the skin layer below the stratum corneum, which is the target site for hyperchromia treatment. Furthermore, the developed microemulsion was shown to be non-irritating to the skin, constituting a safe formulation for topical cutaneous application. Finally, all the promising findings regarding the extract and the formulation potential was confirmed in 3D pigmented skin models, which attested that the proposed loaded EXT microemulsion produced an improved depigmenting effect compared to a commercial formulation. Hence, the developed microemulsion incorporating the cutite fruit extract is a promising novel formulation for treating hyperpigmentation conditions such as melasma.Pouteria macrophylla) (Lam.) Eyma em nanoemulsão e seu uso tópico despigmentante para tratamento de hipercromias. Depositante: Fundação Universidade de Brasília, Universidade Federal do Pará. BR 10 2021 008059 0. Depósito: 27 abr. 2021.GRATIERI, T. et al. Composição contendo extrato do fruto do Cutite (


Pouco se sabe sobre as manifestações clínicas e a evolução do quadro neste grupo de pacientes.No final de 2019, a infecção por um novo coronavírus, causando síndrome respiratória aguda grave, foi relatada em Wuhan, na China.Caso 1: sexo masculino, 47 anos, branco, portador de doença de Chagas, transplante cardíaco em 08/08/2012, sem comorbidades, em uso de tacrolimus (4 mg/dia), micofenolato de sódio (1.440 mg/dia).2: 93%). A gasometria arterial mostrou hipóxia leve. Os demais exames são apresentados na 2), com febre e piora da imagem radiológica, com consolidação em hemitórax direito, e intensificação da opacificação em vidro fosco em hemitórax esquerdo  procurou atendimento de urgência. Exame físico: febril (T. axilar: 38ºC), sem instabilidade hemodinâmica , saturação limítrofe de oxigênio  e micofenolato de mofetil (1000 mg/dia).2: 94%), sem dispneia. A gasometria arterial estava normal; os demais exames são apresentados na 2), sendo o último dia com febre. O ecocardiograma mostrou função sistólica biventricular preservada . No 6º dia de hospitalização (D9 dos sintomas) a tomografia computadorizada evidenciou melhora da imagem radiográfica, acometendo menos de <50% do parênquima pulmonar (No dia 11/05/2020 apresentou febre (38 ºC), tosse seca, falta de apetite e astenia. Sua filha tinha os mesmos sintomas. Em 13/05/2020 (D3 dos sintomas) procurou o pronto-socorro, e o exame físico não evidenciou febre ou instabilidade hemodinâmica ; saturação de oxigênio , sodium mycophenolate (1440 mg/day).2: 93%). Arterial blood gases showed mild hypoxia. Other exams are shown in rd day of hospitalization (D6 of symptoms), the patient remained clinically stable (94% SatO2), with fever and worsening of the radiological image, with consolidation in the right hemithorax, and intensification of the ground glass opacification in the left hemithorax , the fever persisted. A CT scan showed disease progression, with impairment of <50% of the lung parenchyma . On the 6th day of hospitalization, leukopenia/lymphopenia/thrombocytopenia improved, and mycophenolate sodium was reintroduced. On this date, an increase in D-dimer levels was observed (<500 ug/L to 2.270 ug/L). Vascular Doppler of lower limbs did not show any alterations. He was discharged on the 12th day of hospitalization, being asymptomatic. Laboratory tests are shown in On 04/12/2020, he had myalgia, fever, headache, mild dyspnea, dry cough, diarrhea and anosmia. He lives in a community with confirmed cases of COVID-19. On 04/14/2020 (D3 of symptoms) he sought emergency care. Physical examination: feverish (axillary T: 38ºC), without hemodynamic instability , borderline oxygen saturation  and mofetil mycophenolate (1000 mg/day).2: 94%), without dyspnea. Arterial blood gases were normal; other exams are shown in rd day of hospitalization (D6 of symptoms), he was clinically stable, (95% SatO2), and that was the last day he had fever. The echocardiogram showed preserved biventricular systolic function (Teichholz EF: 70.4). On the 6th day of hospitalization (D9 of symptoms) the CT scan showed improved radiographic image, affecting less than <50% of the lung parenchyma , dry cough, lack of appetite and asthenia. His daughter had the same symptoms. On 05/13/2020 (D3 of symptoms) he sought emergency care, and the physical examination showed no fever or hemodynamic instability ; oxygen saturation (SpOrenchyma . On 05/14 In these cases, the clinical presentation was moderate and similar to that reported in non-immunosuppressed patients.4 The inconclusive results of the RT-PCR SARS-COV2 test of the nasopharyngeal swab in Case 2 is in agreement with studies showing that only 63% of the tests performed were positive.5 Although there are no criteria for hospitalization, according to the recommendations from the WHO and the Brazilian Ministry of Health, we chose to indicate hospitalization, as respiratory viral diseases represent a significant cause of morbidity and mortality in immunocompromised patients.7 Case 1 remained feverish until the 10th day of illness, with leukopenia, severe lymphopenia and thrombocytopenia, which made us suspend the use of mycophenolate sodium. It was not necessary to suspend the immunosuppressive regimen of Case 2. Despite the radiological image and inflammatory markers, there was no ventilatory worsening or hypoxemia during the course of the disease. Tomographic images were compatible with the diagnosis, and in Case 2, clinical and radiological parameters allowed the diagnosis to be attained.8 Studies demonstrate immunological participation in the etiopathogenesis of coagulation disorders associated with COVID-19.9 The patients showed D-dimer elevation, but in Case 1 this increase occurred later, on the 11th day of illness. They used prophylaxis for thrombosis, and there were no thromboembolic events. There is currently no proven specific therapy available for COVID-19.11 The treatment follows indications similar to that in non-transplant patients. The hydroxychloroquine regimen was used based on in vitro effectiveness and clinical data on treatment for COVID-19 in the first patient, but not in the second. This change occurred because on this date, studies have not shown evidence of great effectiveness of this drug in the treatment of COVID-19.11 Azithromycin and Ceftriaxone were used for the possible association with bacterial pneumonia.Immunosuppression has been described as a risk factor for greater severity in COVID-19 infection, as well as advanced age, cardiovascular diseases, diabetes mellitus, neoplasms and chronic lung diseases.Changes in the immunosuppressive regimen must be analyzed individually, according to the evolution of each case. The clinical evolution was not more severe than that observed in non-immunosuppressed patients. Studies are needed to assess whether the use of immunomodulators could mitigate the inflammatory cascade. In these specific cases, no exuberant inflammation was observed, which could be associated with the chronic use of immunosuppressants.