Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog’s rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as  R)-3,5-bis (trifluoromethyl)-1-phenylethanol is an important synthon for the synthesis of Aprepitant [R)-4-cyano-3-hydroxybutyric acid is an important intermediate for the preparation of the cholesterol-lowering drug, Atorvastatin [Enantiomerically pure chiral alcohols are pivotal building blocks for the production of chiral pharmaceuticals, agrochemicals, flavors and other fine chemicals with chiral centers ,2,3,4. Frepitant ,8. Ethylvastatin . Currentvastatin ,11,12,13vastatin ,15.Rhodotorula sp. AS2.2241 was used as a whole-cell biocatalyst in the synthesis of enantiopure (S)-1-(4-methoxyphenyl) ethanol from asymmetric reduction of 4′-methoxyacetophenone with 99% ee [Rhodotorula sp. AS2.2241 cells showed improved stability and solvent resistance in biocatalysis [ee towards structurally related aromatic ketones were achieved by some of the examined marine fungi [Biocatalytic asymmetric reduction of prochiral ketones for chiral alcohols can be performed by using isolated enzymes and whole cells. Compared with enzymatic processes, microbial whole cell catalyzed reactions are preferable and are often well accepted for practical reasons ,17,18. Uh 99% ee . The immatalysis . Recentlne fungi . Althougne fungi ,29. Ketone fungi . Howeverne fungi . Thus, tBacillus cereus TQ-2 with good enantioselectivity for the biocatalytic reduction of prochiral ketones for the production of chiral alcohols. The resting cells of TQ-2 exhibit high activity and excellent anti-Prelog enantioselectivity toward the model substrate, acetophenone. The catalytic properties of TQ-2 whole cells and the key factors of the reaction conditions were investigated in detail. In addition, the cells were highly active to a panel of structurally diverse ketones and aldehydes, indicating a broad substrate spectrum and good catalytic activity as a new whole-cell biocatalyst.In this study, we report a new isolated bacterial strain of ee values ranging from 78% to 99% (R)-1-phenylethanol as the sole product, exhibiting the best conversion and excellent enantioselectivity , having sequence identities of 99% -5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester, an important chiral intermediate of indoxacarb synthesis [Bacillus cereus was reported as whole cell biocatalyst for use in the production of anti-Prelog chiral alcohols from prochiral ketones. The discovery of B. cereus TQ-2 with anti-Prelog stereopreference is of significance not only for meeting the demands of chiral alcohol synthesis, but also for exploring the distribution of microorganisms that can be used in the bioreduction of ketones following anti-Prelog’s rule in nature.Currently, a number of anti-Prelog specific whole cell biocatalysts have been discovered in different microbial species, including JUY-1401 , AcetobaGIM1.158 , Acetoba M209061 , Torulasii 20126 , TrichodZJPH0810 , Lactobaus kefir , Geotricpolytica , Lactobas brevis  and Oenocus oeni . Severalcus oeni ,31. The gh yield . Zhang eynthesis . HoweverB. cereus TQ-2 cells showed considerable activity at a broad temperature range from 20 °C to 45 °C. The best acetophenone conversion of 39.8% was achieved at 30 °C. Meanwhile, a conversion of 21.8% was obtained at 45 °C. In addition, by increasing the reaction time to 48 h, the conversion was improved slightly, reaching 49.1%. Nevertheless, the concentration of (R)-1-phenylethanol product in the reaction system decreased slightly rather than increased over reaction time , and further increases did not improve the conversion. The conversion decreased to 54% in the presence of 25% (v/v) glycerol. A higher proportion of glycerol may lead to higher viscosity, which could impact the mass transfer of the reaction mixture. Therefore, 15% (v/v) of glycerol was selected in the subsequent experiments.Furthermore, the optimal glycerol concentration for an acetophenone reduction catalyzed by termined b. It wasB. cereus TQ-2 whole-cell biocatalyst was examined. The metal ions were added to the reaction mixture with a final concentration of 1.0 mM. As shown in 2+ or K+ ions significantly enhanced the conversion from 48.9% to 59.7% and 61%, respectively. However, the addition of 1.0 mM of Cd2+ dramatically decreased the conversion to 15.3%, which is in agreement with previous reports that demonstrated the negative effect of heavy metal ions on enzyme activity. Moreover, the catalytic activity of TQ-2 whole cells was completely inhibited by the addition of 1.0 mM of Cu2+. Nevertheless, another heavy metal ion, Ni+, did not have a significant influence on the conversion. A slight decrease in conversion was observed in the presence of 1.0 mM of Mn2+, Mg2+ or Fe2+ ions. It has been reported that many of the alcohol dehydrogenase catalyzing selective reductions of prochiral ketones to enantiopure alcohols are zinc-containing enzymes. However, in this experiment, adding Zn2+ ions into the reaction mixture did not improve the conversion, implying that the possible reductase in the TQ-2 cells may not be zinc-dependent.It is well established that metal ions play an important role in maintaining the intact structure of some enzymes, which is a prerequisite for their catalytic activities . Thus, tR)-1-phenylethanol from B. cereus TQ-2 whole cell catalyzed selective reduction of acetophenone was successfully obtained, providing a yield of 52% (isolated yield). In addition, in order to expand the applications of the newly discovered B. cereus TQ-2 for biocatalytic reductions, the biocatalytic potential of the cells for the synthesis of various alcohols in selective carbonyl compound reductions was explored. As known, alcohols, especially chiral alcohols, are often used as important building blocks in organic synthesis and pharmaceutical applications. In this study, a set of structurally different ketones and aldehydes was used in reduction reactions. As shown in B. cereus TQ-2 cells exhibited considerable activity towards the tested compounds with different structures, leading to corresponding alcohol products without byproducts. Compounds 1a–1k were converted to corresponding chiral alcohol products with a 30% to 100% conversion, yielding modest to excellent stereoselectivity.Under the optimized reaction conditions, a  and ee values . Interestingly, excellent selectivity and conversion were achieved for compounds 1h and 1i, yielding valuable enantiomerically pure R-selective alcohols with 99% ee. However, the substitution on the ortho-position or meta-position of acetophenone resulted in no conversions during the asymmetric reductions . It seemed that the substituents at the ortho-position and/or meta-position severely prevented the occurrence of a reaction. A similar phenomenon was reported for a newly isolated ChKRED20 from Chryseobacterium sp. CA49 [B.cereus TQ-2 cells would provide a good explanation of the catalytic reactions at the molecular level. Moreover, the strain exhibited excellent chemoselective activity towards structurally different aliphatic and aromatic aldehydes in reduction reactions, achieving a >95% conversion, except for 1l. The bioreduction of aldehyde by B. cereus TQ-2 provides a useful alternative to chemical methods for the production of primary alcohols which can be further oxidized to their respective carboxylic acids.The activity and stereoselectivity of sp. CA49 . The isoR)-(−)-α-(Trifluoromethyl) benzyl alcohol, (R)-(+)-1-Phenyl-1-butanol, and (R)-(−)-4-Phenyl-2-butanol were purchased from Sigma Aldrich Trading Co., Ltd. . NADH and NADPH disodium salt were purchased from Aladdin Bio-Chem Technology Co., Ltd.  and Bide Pharmatech Ltd. , respectively. LB medium and the other media for bacteria enrichment and cultivation were prepared following standard protocols.Acetophenone, acetophenone derivatives and the other chemical reagents involved in this study were purchased from commercial suppliers and used directly without further purification. (v/v) of glycerol.Soil samples collected from the forest of Wutong Mountain located in Shenzhen City in the Guangdong Province of China were used to screen for microorganisms with reduction activity to the model substrate of acetophenone. The bacteria were isolated following a conventional enrichment cultivation procedure. Mineral salt medium (MSM) supplemented with 1 g/L of acetophenone was used for the subsequent enrichment experiments. The entire enrichment process followed the protocol reported by Li et al., with slight modification . Namely,The isolated bacteria were grown at 30 °C, 200 rpm for 20 h in LB medium composed of tryptone 10.0 g/L, yeast extract 5.0 g/L, NaCl 10.0 g/L. The cells of bacteria were harvested and examined for the reduction activity using acetophenone as substrate following the procedure described in B. cereus TQ-2 was deposited in the Genbank database, with the accession number OP035935.The isolated strains with good catalytic performance in transforming acetophenone into the corresponding alcohol were submitted to the China Center Type Culture Collection (CCTCC) for identification based on their physiological and biochemical characters and 16S rDNA sequence. The Bacillus cereus TQ-2 strain was deposited in CCTCC  with the accession number CTCC JD 2019287. Phylogenetic trees of the aligned sequences and the closest 16S rDNA matches were constructed using a neighbor-joining method using MEGA 5 with 1000 bootstrap replicates. The 16S rDNA sequence of B. cereus TQ-2 was inoculated into 5 mL of LB liquid media for overnight cultivation at 30 °C with shanking at 200 rpm. Then, 1 mL of the overnight-cultivated culture was added to 100 mL of fresh LB liquid media for cultivation under the same growth conditions. After 24 h, cell pellets were collected by centrifugation and washing twice with 100 mM, pH 7.4, phosphate buffer.The glycerol stock of v/v) acetonitrile as co-solvent. After incubation at 30 °C and 600 rpm for 24 h, the transformation was terminated by adding 2 mL of ethyl acetate into the reaction mixture. The organic phase was extracted and filtered for GC analysis.The biotransformation of acetophenone and other carbonyl substrates was carried out in a 50-mL screw-capped Falcon centrifuge tube. The reaction mixture was prepared as follows: 5 mL of phosphate buffer  contained 0.5 g of wet cells and 2 mM of acetophenone or other carbonyl substrates with 1% (1l): Injector 250 °C, Detector 250 °C; 80 °C (1 min) 6 °C/min, 120 °C (1 min). Program B : Injector 270 °C, Detector 300 °C; 60 °C (4.5 min), 20 °C/min, 80 °C (1 min) 20 °C/min, 280 °C (5 min).The products of the reactions were measured using the Agilent 8860 GC system instrument with a flame ionization detector. The product identification was performed through comparison with authentic compounds from commercial suppliers and analyzed by GC chromatography. The conversions of substrates were measured with an HP-5MS column (30 m × 0.32 mm × 0.25 μm) using the following programs: Program A : Injector 220 °C, Detector 220 °C; 80 °C (0.5 min), 10 °C/min, 110 °C (5 min), 10 °C/min, 130 °C (6 min), 25 °C/min, 180 °C (0.5 min). Program D (1g): Injector 220 °C, Detector 220 °C; 80 °C (1 min), 10 °C/min, 110 °C (5 min), 10 °C/min, 130 °C (6 min), 25 °C/min, 180 °C (5 min), 25 °C/min, 200 °C (1 min). Program E : Injector 230 °C, Detector 230 °C; 80 °C (1 min), 10 °C/min, 110 °C (5 min), 10 °C/min, 130 °C (6 min), 10 °C/min, 180 °C (5 min), 25 °C/min, 200 °C (4 min).The separation of enantiomers of chiral alcohols was performed using a chiral Cyclodex-B column (30 m × 0.25 mm × 0.25 μm) using the following programs: Program C . Injector 250 °C, Detector 250 °C; 90 °C (5 min), 3 °C/min, 105 °C, 5 °C/min, 120 °C, 20 °C/min, 180 °C.The transformation of 1k transformation was performed on a Chiralcel OD-H column  using n-heptane: isopropanol  as a mobile phase at a flow rate of 1.0 mL/min at 25 °C and monitored with a detector at 254 nm. The absolute configuration of the alcohol products was determined through the comparison of their retention time with the related literature or authentic compounds obtained from commercial sources. All of the experiments were performed in triplicate and the mean values are presented.The analysis of B. cereus TQ-2 cells are now possible in future studies. This is the first report of a strain from B. cereus species with anti-Prelog stereoselectivity for use in prochiral ketone reduction. Our study provides a whole-cell biocatalyst that can be used in asymmetric ketone reduction, setting the stage for future work concerning the isolation and identification of related enzymes.Herein, Bacillus cereus TQ-2, a new whole-cell biocatalyst with excellent anti-Prelog stereoselectivity for the asymmetric reduction of prochiral ketones was successfully isolated from a soil sample. The resting cells of the strain showed considerable activity over a broad temperature range and remarkable pH adaptability. Moreover, it was found that the cells exhibit a broad substrate spectrum and can utilize glycerol as a co-substrate for coenzyme regeneration. Thus, many applications of


CTSS, CAT, CNDP1, KNG1, SLAMF7, TIE1, CXCL1, MBL2, ESD, CXCL16, CCL14, KCNE5, CST7, PSME1, GPC3, MAP2K4, SPOCK3, LRPPRC, CLEC4M, NOG, C1QTNF9, CX3CL1, SCP2D1, SERPINF2, and FN1). These proteins were enriched in biological processes such as regulation of peptidase activity and regulation of cellular protein metabolic processes. Proteins  involved in the Ras signaling pathway were identified to be related to AAD. In addition, SCAD- and AAD-associated SNPs were associated with cytokine and lipid levels. MR analysis showed that circulating ECM1, SPOCK3 and IL1b levels were associated with AAD. Circulating levels of low-density lipoprotein cholesterol and small very-low-density lipoprotein particles were strongly associated with AAD. The present study found associations between circulating proteins and lipids and SCAD and AAD. Circulating ECM1 and low-density lipoprotein cholesterol may play a role in the pathology of SCAD and AAD.Circulating proteins play functional roles in various biological processes and disease pathogenesis. The aim of this study was to highlight circulating proteins associated with aortic aneurysm and dissection (AAD) and spontaneous coronary artery dissection (SCAD). We examined the associations of circulating molecule levels with SCAD by integrating data from a genome-wide association study (GWAS) of CanSCAD and 7 pQTL studies. Mendelian randomization (MR) analysis was applied to examine the associations between circulating molecule levels and AAD by using data from UK Biobank GWAS and pQTL studies. The SCAD-associated SNPs in 1q21.2 were strongly associated with circulating levels of extracellular matrix protein 1 (ECM1) and 25 other proteins (encoded by  Aortic aneurysm and dissection (AAD), characterized by intimal rupture and intramural hematoma formation, is a life-threatening disease that affects the aorta. AAD is associated with a high risk of mortality . With thCOL3A1  is a rare event with an incidence of approximately 0.28–1.1% and occurs most commonly in women . In SCADCirculating proteins play functional roles in various biological processes and disease pathogenesis and have been suggested to be druggable targets , 22. RecCirculating lipid composition plays a key role in AAD . The assP-values of 607,778 genotyped variants with SCAD (GCST90000582). The dataset contains six columns, which present the information of rs ID, chromosome, position, effect allele, non-effect allele and P-value for each SNP. SNPs with P < 1.0 × 10–5 were identified, and the SCAD loci were determined. The genome-wide significance threshold was set to 5.0 × 10–8 in this study.In this study, we determined genetic loci for SCAD according to the GWAS from the Canadian SCAD (CanSCAD) study , which cith SCAD , which c1 The summary data for AAD can be found by searching “Aortic aneurysm and dissection” at this website. The downloaded dataset contains information on SNP ID, effect allele, non-effect allele, allele frequency, beta, standard error, and p-value for each SNP, which are necessary for the MR analysis. SNPs with minor allele frequencies greater than 0.05 in this dataset were used. These summary data were used in the MR analysis in this study.GWAS summary data of AAD (ICD-10 code: I71), which contains 452,264 individuals from the UK Biobank, were obtained. GWASs for 778 traits in the UK Biobank were performed . The GWAP-values less than 1.0 × 10–5 were considered in this study. The pQTL analyses were performed in the five pQTL studies listed below. The downloaded datasets contain information on SNP ID, effect allele, non-effect allele, allele frequency, beta, standard error and p-value for each SNP, which are necessary for the MR analysis. Based on these pQTL data, circulating proteins associated with the SCAD- and AAD-associated SNPs were identified, and MR analyses were performed.We carried out pQTL analysis in peripheral blood for the identified SCAD- and AAD-associated SNPs to identify their functional potentials. pQTL signals with http://metabolomics.helmholtz-muenchen.de/pgwas/index.php?task=download. The data contain associations between 509,946 SNPs and 1,124 proteins.(i) The KORA study. This study quantified levels of 1,124 proteins in blood plasma samples of 1,000 individuals. Associations between genome-wide SNPs and the protein levels were examined; that is, 1,124 GWASs were performed  The INTERVAL pQTL study.AL study .3 This pQTL study is a GWAS that analyzed genome-wide associations between SNPs and 83 proteins considered relevant to cardiovascular disease measured in 3,394 individuals ((iii) The cardiovascular disease-relevant protein GWAS.ividuals .(iv) The Framingham Heart Study. This pQTL study published 16,602 pQTL results for 71 high-value cardiovascular disease proteins that were measured in 7,333 Framingham Heart Study participants .4(v) The AGES Reykjavik study. A total of 1,046 significant pQTL results were obtained from this pQTL study. This study measured 4,137 proteins on the Slow-Off rate Modified Aptamer (SOMAmer)-based proteomic profiling platform (SOMAscan) in 5,457 Icelanders . AssociaStudies of the link between cytokines and growth factors, which are regulators of inflammation, and AAD could deepen our understanding of the disease and may guide future treatment therapies. Therefore, we searched for links between AAD-SNPs and cytokine levels. A pQTL study examined the genome-wide associations between 10.7 million SNPs and 41 cytokine concentrations in up to 8,293 Finnish individuals . Based oBlood metabolites could potentially serve as biomarkers for SCAD and AAD. Comprehensive molecular profiling of circulating metabolites can help to uncover the metabolic pathophysiology underlying SCAD- and AAD-associated variants. We identified associations between SCAD- and AAD-SNPs and circulating metabolites. A total of 123 metabolites that represent a broad molecular signature of systemic metabolism in blood were quantified using the quantitative high-throughput nuclear magnetic resonance (NMR) metabolomics platform in up to 24,925 individuals. Associations between 39 million genetic markers and the metabolite concentrations were examined and the summary data are publicly available .5 was used to carry out the GO analysis and illustrate the functional enrichments (We then carried out gene ontology (GO) analysis. GO contains three independent ontologies: biological process, molecular function and cellular component . GO analichments .For the identified proteins, we performed MR analyses based on the GWAS summary data to test if circulating levels of the molecules identified in the pQTL studies were associated with AAD. The association between circulating protein levels and SCAD was not examined because some necessary data  are unavailable. The weighted median , inverseP-values) were extracted from the summary datasets of GWAS and pQTL studies described above. In the pQTL summary data, SNPs with a P-value less than 1.0 × 10–5 were selected as potential instrumental variables. The selection criterion was not set to 5.0 × 10–8 because this stringent threshold would lead to too few instrumental variables, which may hinder the analyses. We clumped SNPs  based on data from Europeans from the 1,000 Genomes project using the “clump_data” function in the R package TwoSampleMR to select independent instrumental variables. The consistency of the effect allele for each SNP in the BP GWAS and pQTL studies was checked and the direction of the effect was corrected when inconsistency existed. The IVW MR and MR–Egger analyses were performed by using the MendelianRandomization R package  GWAS, which identified more than one hundred lipid-associated loci, were applied in the present study , 43. Thi6-methyladenosine (m6A)-associated SNPs6 (7 (phosSNPs)   and SNPsSNPs) . The top SNP associated with SCAD in 1q21.2 was rs12740679 (P = 2.19 × 10–12). There were fewer genome-wide significant SNPs in other selected loci. Four SNPs, rs11172113 (P = 2.63 × 10–8), rs10747776 (P = 4.92 × 10–8), rs4367982 (P = 2.77 × 10–8), and rs4759277 (P = 4.77 × 10–8), in 12q13.3 and one SNP  in 6p24.1 were significantly associated with SCAD. For the remaining ten selected loci, no SNP reached the genome-wide significance level. For 2p23.2, 4q34.3, 5q23.2, and 12q21.33, only one SNP reached 1.0 × 10–5 in each locus. In 6q25.3, 11 SNPs passed 1.0 × 10–5. The top SNP in 6q25.3 was rs78349783 (1.03 × 10–6). SNPs in these 13 loci were considered in the following analysis.According to the SCAD GWAS summary data, we picked up 13 SCAD-associated loci, including 1p32.1, 1q21.2, 2p23.2, 2q33.2, 4q34.3, 5q23.2, 6p24.1, 6q22.1, 6q25.3, 12q13.3, 12q21.33, 12q23.2, and 21q22.11 . The top–5 were identified from the UK Biobank dataset, and 4 SNPs  in the OLFML1 gene region (11p15.4) passed 5.0 × 10–8. There were 50 SNPs nominally associated with AAD in this locus and the top SNP was rs112809417 (P = 4.10 × 10–8) (–6) and rs1417488 (5.05 × 10–6) were found in the TGFB2 gene  . SNPs inFB2 gene .P = 7.73 × 10–102), followed by rs832622 (P = 3.91 × 10–95) . Among the genome-wide significant SCAD-associated SNPs, rs698915, rs9126, and rs3850844 were significantly associated with circulating levels of ECM1 (P = 1.45 × 10–6) and circulating levels of SERPINF2 (P = 2.69 × 10–10), FN1 (P = 6.24 × 10–15), and ECM1 (P = 1.01 × 10–45)  . However of ECM1 . In anot× 10–45) .P < 0.05) associated with SCAD, were associated with 637 proteins (CA6 gene) at 1p36.23 was strongly associated with the circulating level of CA6 (P = 8.70 × 10–113); rs4584384 (intronic SNP in the TDRD10 gene) at 1q21.3 was strongly associated with circulating levels of IL6R (P = 1.84 × 10–33).In addition to the genome-wide significant SNPs, a total of 184,983 SNPs, which were nominally (proteins . On chroproteins . For exaP = 1.10 × 10–5), secretory granule lumen (P = 9.80 × 10–5), and extracellular exosome (P = 1.20 × 10–4); biological processes such as regulation of peptidase activity (P = 1.70 × 10–5) and movement of cell or subcellular component (P = 2.90 × 10–5); and molecular functions such as chemokine activity (P = 3.90 × 10–4) and chemokine receptor binding (P = 8.10 × 10–4)  were affected by genetic variants at 1q21.2. These proteins were enriched in biological processes such as regulation of peptidase activity (P = 9.40 × 10–7), regulation of cellular protein metabolic process (P = 4.10 ×10–6), regulation of protein metabolic process (P = 2.20 × 10–5); molecular function such as peptidase regulator activity (P = 5.10 × 10–7), and chemokine activity (P = 4.50 × 10–5); cellular component such as secretory granule lumen (P = 1.90 × 10–4) and cytoplasmic membrane-bounded vesicle lumen (P = 3.40 × 10–4), and so on , including cellular components such as vesicle lumen ( × 10–4) . Concentnd so on .OLFML1 were associated with circulating levels of SLURP1, PLAU, CDKN1B, ACVR1B, and STAB2. Furthermore, we performed MR analysis to evaluate the associations between the identified proteins and AAD. Circulating ECM1 levels were nominally associated with AAD in IVW (P = 0.0129) and MR-PRESSO (P = 0.0193) analyses. Circulating SPOCK3 levels were associated with AAD in weighted median (P = 0.0024), IVW (P = 0.0013), and MR-PRESSO (P = 6.94 × 10–4) analyses.A total of 64 proteins were found to be associated with AAD-associated SNPs. Proteins  involved in the Ras signaling pathway were identified. The genome-wide significant SNPs in P < 0.05), we found 220,594 SNPs that were nominally associated with circulating levels of 42 cytokines. For SNPs associated with SCAD at P < 1.0 × 10–5, we found that rs1631973 (SCAD association P = 1.49 × 10–6) and rs9436119 (SCAD association P = 1.52 × 10–6) were associated with cytokine bNGF levels . In addition, rs7764227 (SCAD association P = 5.34 × 10–3) and rs9369433 (SCAD association P = 5.82 × 10–3) were associated with circulating levels of IL10 , IL12p70 , IL13 , IL7 , and VEGF .For SNPs nominally associated with SCAD  and rs141292109 (AAD association P = 1.56 × 10–7) were associated with IL7 . A total of 28 SNPs were nominally associated with the IL1b level (P < 0.05). In the MR analysis, the IL1b level was nominally associated with AAD in the weighted median (P = 0.0031), IVW (P = 0.0092), MR–Egger (P = 0.0422), and MR-PRESSO (P = 0.0165) analyses.For SNPs associated with AAD at P < 0.05), we found 48,689 SNPs that were nominally associated with circulating levels of the 123 circulating metabolic traits (P = 8.92 × 10–3), rs6717111 (SCAD association P = 6.08 × 10–3), rs4310999 (SCAD association P = 8.63 × 10–3), rs13029304 (SCAD association P = 6.86 × 10–3), rs4630697 (SCAD association P = 6.98 × 10–3),and Gly , followed by the associations between rs174603 (SCAD association P = 7.66 × 10–3) and rs11230815 (SCAD association P = 1.03 × 10–3) and otPUFAs . For SNPs associated with SCAD with P < 1.0 × 10–5, no SNP was associated with metabolites at P < 1.0 × 10–5, but there were 329 SNPs associated with 85 metabolites at P < 0.05 (P < 1.0 × 10–7) (P = 1.44 × 10–9) was strongly associated with circulating levels of LDL-D (P = 3.99 × 10–5). In addition to rs7366347, 281 SNPs (SCAD association P < 1.0 × 10–5) at 1q21.2 were associated with circulating levels of LDL-D (P < 0.05).For SNPs nominally associated with SCAD (c traits . The topP < 0.05 . These r × 10–7) . The SCAP < 5.0 × 10–5, no SNP was associated with metabolites at P < 1.0 × 10–5. However, for SNPs nominally associated with AAD, more than 500 association signals were found for metabolites such as L.HDL.P, L.HDL.L, HDL.C, L.HDL.PL, L.HDL.CE, M.VLDL.P, XL.HDL.TG, XS.VLDL.L, APOB, XS.VLDL.P, S.VLDL.FC, L.HDL.C, S.VLDL.C, L.HDL.FC, L.VLDL.CE, IDL.TG, M.VLDL.TG, and XS.VLDL.TG.Associations between AAD-associated SNPs and 122 metabolic traits were found. The top signal was found for otPUFA. For SNPs associated with AAD at P = 7.80 × 10–3), IVW (P = 1.17 × 10–4), and MR-PRESSO (P = 1.94 × 10–4) analyses (P = 1.21 × 10–3), MR–Egger (P = 2.63 × 10–3), and MR-PRESSO (P = 3.97 × 10–4) analyses. HDL-c and TG were found to be associated with AAD in the weighted median , IVW , and MR-PRESSO  analyses. We also found that high cholesterol and lipidemia were significantly associated with AAD in weighted median , IVW , and MR-PRESSO  analyses, and nominal associations in MR–Egger  analyses were found. We performed a multivariable MR analysis for the association between circulating levels of LDL-c, HDL-c, TC and TG and AAD. After adjusting for HDL-c or TG, LDL-c was associated with AAD . When adjusted for TC, the association between LDL-c and AAD was not significant.In the MR analysis, LDL-c levels were associated with AAD in the weighted median , IVW (P = 8.92 × 10–4) and MR-PRESSO (P = 1.34 × 10–3) analyses. In addition, total lipids in small very-low-density lipoprotein particles (S.VLDL.L) level was significantly associated with AAD  , S.VLDL.FC , S.VLDL.PL , and XS.VLDL.TG  were also found to be associated with AAD. The association between S.VLDL.L (P = 9.05 × 10–5), S.VLDL.C (P = 1.48 × 10–3), S.VLDL.FC (P = 2.49 × 10–4), S.VLDL.PL (P = 1.96 × 10–3), XS.VLDL.TG (P = 4.01 × 10–4) and AAD were significant in MR-PRESSO analyses. In multivariable MR analysis, the association between circulating levels of S.VLDL.L and AAD was not significant after adjusting for S.VLDL.FC or XS.VLDL.TG.Circulating levels of otPUFA were associated with AAD in weighted median ( × 10–3) . Other vRPRD2 and rs3818978 (5′-UTR) in MRPS21 were associated with m6A methylation. The SNP rs12758270 was significantly associated with ECM1 mRNA expression levels in fibroblast cells (P = 2.79 × 10–5) and rs3818978 was significantly associated with ADAMTSL4 and MRPS21 gene expression levels in aortic artery tissue . The SNP rs580060 (3′-UTR) in MRPS21 was associated with A-to-I RNA methylation and ADAMTSL4 mRNA levels in aortic artery tissue (P = 1.27 × 10–6).We searched for several kinds of functional variants among pQTLs that may affect circulating proteins and metabolites in public databases. We found that rs12758270 (3′-UTR) in ECM1 were associated with ADAMTSL4 mRNA levels in aortic artery tissue  and may have impacts on the phosphorylation of ECM1 protein.The missense variants rs3737240 [T (ACG) > M (ATG)] in the sixth exon and rs13294 [G (GGT) > S (AGT)] in the eighth exon of ALK gene variant rs4535004 (P = 2.60 × 10–4) and the RPRD2 gene variants rs7002  and rs834230 (P = 2.62 × 10–4) altered the activity of putative regulatory elements in K562 cells. The RPRD2 gene variant rs11205370 (P = 8.08 × 10–4) altered the activity of putative regulatory elements in HepG2 cells. Among these four regulatory SNPs, rs7002 (P = 1.16 × 10–6), rs834230 (P = 1.83 × 10–6), and rs11205370 (P = 8.23 × 10–7) were associated with ADAMTSL4 mRNA levels, and rs11205370 (P = 5.35 × 10–7) was associated with MRPS21 gene expression levels in aortic artery tissue.The This study searched for potential molecules that were related to SCAD and AAD by integrating data from GWAS and pQTL studies. We found proteins, cytokines and metabolites for SCAD and AAD and highlighted pQTLs at 1q21.2 that were strongly associated with circulating levels of ECM1. Moreover, this study also found potential causal proteins and lipid metabolites for AAD.ECM1 gene is located at chromosome 1q21. This gene encodes the ECM1 protein, a widely expressed 85-kDa glycoprotein  in MRPS21, have the potential to affect m6A methylation in the mRNA of RPRD2 and MRPS21. The associations between rs12758270 and ECM1 mRNA expression levels in fibroblast cells and between rs3818978 and ADAMTSL4 and MRPS21 gene expression levels in aortic artery tissue support their potential functions. The SNP rs580060 in the 3′-UTR of MRPS21 may affect A-to-I RNA methylation and was associated with ADAMTSL4 mRNA levels in aortic artery tissue. In addition, missense variants rs3737240 and rs13294 in ECM1 were associated with ADAMTSL4 mRNA levels in aortic artery tissue. Moreover, rs3737240 and rs13294 are phosphorylation-associated SNPs that may have impacts on the phosphorylation of ECM1 protein, and rs13294 was found to be associated with circulating ECM1 levels. These functional SNPs may alter protein levels and subsequently affect SCAD and AAD risk. However, how these SNPs participate in the pathogenesis of SCAD and AAD by altering circulating proteins needs to be elucidated in future studies.We identified many putative functional SNPs among the pQTLs. Two SNPs, rs12758270 (3’-UTR) in This study showed that SCAD-associated and AAD-associated SNPs may have strong effects on circulating levels of proteins and metabolites. Therefore, this study found circulating proteins and metabolites that were pleiotropically associated with AAD. Circulating ECM1 and LDL-c may play important roles in the pathology of AAD. Genetic variants at 1q21.2 may have regulatory potential to affect gene expression in related tissues, such as aortic artery and circulating protein levels, and subsequently affect SCAD and AAD risk. This study increased our understanding of the regulatory roles of genetic variants in SCAD and AAD susceptibility genes. The findings highlighted potential pathways for further detection of functional mechanisms underlying the associations between genetic variants and SCAD and AAD.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Fujian Medical University. The patients/participants provided their written informed consent to participate in this study.TC and LC designed this study. TC, XY, and ZQ collected the data. TC was responsible for the statistical analysis. MT and TC wrote the draft. MT, XL, and LC revised this draft. LC finalized this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We focused on the functional components, antioxidant activity, skin-whitening, and anti-wrinkle properties of subcritical and supercritical water (SCW)-treated rutin. Rutin treatments were performed at the following temperature and pressure conditions: 200 °C/15 bar, 300 °C/100 bar, and 400 °C/250 bar. ABTS and DPPH radical scavenging activities and reducing power presented their highest values  at 300 °C/100 bar. The tyrosinase inhibitory activity of SCW-treated rutin was 21.72–60.05% at 1 mg/mL. The ethyl acetate fraction showed 14.91% melanin inhibitory activity at a concentration of 10 µg/mL compared to the α-MSH treatment group. The protein expression inhibition rates of MITF, tyrosinase, TRP-1, and TRP-2 in the ethyl acetate fractions were 14.05%, 72%, 93.05%, and 53.44%, respectively, at a concentration of 10 µg/mL, compared to the control. These results indicate that SCW treatment could be used to develop cosmetic materials and functional food with physiological activity, and that SCW-treated rutin can be used as a skin-whitening cosmetic material. Supercritical water (SCW) refers to water at pressure and temperature above the critical point 218 bar/374 °C), presenting similar properties to solids, liquids, and gases 74 °C, pr,6.l-rhamnopyranosyl-(1-6)-β-d-glucopyranose) are combined. Rutin presents various physiological characteristics, such as antioxidant, anti-diabetic, anti-cancer, and anti-aging. Rutin is known to increase its activity when converted to an aglycone, quercetin, and deglycosylation occurs by various methods, such as chemical treatment, acid treatment, and heat treatment -2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), α-MSH, radio-immunoprecipitation assay buffer (RIPA buffer), protease and phosphatase single-use inhibitor cocktail 100X, and the bicinchoninic acid (BCA) kit were purchased from Sigma Chemicals Co .Subcritical and SCW treatments of rutin were performed using a SCW reactor , reaching temperatures up to 400 °C with maximum pressure up to 300 bar. The pressure of the SCW reactor was set depending on the amount and temperature of water placed in the inner container. Three temperature/pressure conditions were tested: 200 °C/15 bar, 300 °C/100 bar , and 400 °C/250 bar . After reaching the desired temperature, the treatment was conducted for 30 min. A group of untreated samples was used as the control group. In the experiment, 0.3 g of rutin was suspended in 10 mL of distilled water and placed in the inner container, which was then placed in the outer container together with a certain amount of water, and the outer container was completely sealed. Rutin was treated with subcritical and SCW under each condition. The processed rutin was filtered using a 0.45 μm syringe filter , and solvent was removed at 40 °C in a rotary evaporator  and then freeze-dried . The dried sample was used and stored at −80 °C.For solvent fractionation, we used 300 °C/100 bar treated rutin, which had the highest antioxidant and skin whitening activities. After SCW treatment, the sample was suspended in distilled water, and solvent fractionation was sequentially performed using 300 mL (x3) of each solvent in the following order: N-hexane, chloroform, ethyl acetate, butanol, and water ,30. Eachv/v), 2–27 min 10:90, 27–50 min 30:70, 50–51 min 90:10, 51–60 min 100:0, 60–70 min 100:0, and 70–end min 8:92. The sample injection volume was 20 μL, the UV detector was set at 280 nm, and the flow rate was 1 mL/min. Rutin (y = 11361x + 426.15) and quercetin (y = 24495x + 52.046) were purchased from Sigma-Aldrich Co., and the calibration curves of standard materials all showed an R2 value of 0.999 or higher. All samples were analyzed in triplicate.Rutin and quercetin content analyses were conducted using slightly modified methods of Seo et al. and Jung et al. ,32. The The ABTS radical scavenging activities of the SCW-treated rutin and its solvent fractions were measured using the method by Choi et al. . ABTS ra3Fe(CN)6] and 250 μL of 0.2 M sodium phosphate buffer (pH 6.6) were mixed with 250 μL of extract and incubated at 50 °C for 20 min. Then, 1% trichloroacetic acid  was added, and the resulted solution was centrifuged at 1000 rpm for 10 min. Next, 500 μL of distilled water were mixed with 500 μL of the supernatant together with 100 μL of 0.1% ferric chloride (FeCl3∙6H2O), and the absorbance of the solution was analyzed at 700 nm.The reducing power was measured using method by Mau et al. , where 2To determine the skin-whitening effect, the inhibited tyrosinase activity was analyzed using the method by Yagi et al. . In a te4 cells/well in a 96 well plate, and CCD-986sk cells were seeded at 5 × 103 cells/well in a 96 well plate and cultured for 24 h. After inoculation, the sample was diluted and incubated for 24 h, followed by the addition of 40 µL of MTT solution (2.5 mg/mL) and incubation for less than 4 h. After removing the culture medium, 100 µL of DMSO was added to the formazan, generated by the MTT reduction reaction, and incubated at 25 °C for 10 min. The absorbance was measured at 540 nm using an ELISA reader. The cytotoxicity was expressed as a percentage by comparing the sample’s absorbance with that of the control group (sample without any addition).The cytotoxicity was analyzed using the method by Carmichael et al. , where B6 cells/well, cultured for 24 h, and treated with 1 µg/mL of the stimulant α-MSH for 2 h. The sample was then diluted, inoculated, and incubated for 24 h. α-MSH was administered to the single treatment group and a sample section, whereas a section was left without α-MSH treatment and was used as the control group. The cultured plate was washed with PBS twice, and 80 µL of lysis buffer  were added. The resulting pellet was harvested and centrifuged at 13,200 rpm for 20 min. The supernatant was collected, and the protein contents were measured using a BSA kit and a standard curve. After centrifugation, the obtained pellet was dissolved in 10% DMSO mixed with 1 N NaOH solution. After dissolution, the absorbance was measured at 405 nm after the solution was incubated at 90 °C for 1 h using an ELISA reader. The melanin production was calculated and expressed as the percentage of the sample treatment group relative to the control group.The production of melanin was measured using the slightly modified method of Hosoi et al. . B16F10 6 cells/well and cultured for 24 h, after which, they were treated with 1 µg/mL α-MSH for 2 h. Extracts of 2.5, 5, and 10 µg/mL were incubated for 24 h, after which the culture medium was removed, and treatment with phosphate-buffered saline (PBS) followed. After washing twice, 100 µL of protease and phosphatase single-use inhibitor cocktail 100X was added to 10 mL of RIPA buffer, lysed with lysis buffer, and centrifuged at 13,200 rpm at 4 °C for 20 min. The protein content of the supernatant was quantified using a BCA protein assay kit, and 20 µL of protein was separated by electrophoresis using 10% SDS-PAGE. The separated protein was transferred to a polyvinylidekslne fluoride (PVDF) membrane and incubated by blocking for 1 h with 5% skim milk. Primary antibodies against tyrosinase, MITF, TRP-1, and TRP-2 were diluted 1:1000 in 3% skim milk and incubated overnight. After three 10-min washings with tris-buffered saline and Tween 20 (TBST), the secondary anti-mouse antibody was diluted 1:2000, 1:5000, and 1:10,000 in 3% skim milk and reacted at room temperature for 2 h. The washing process was repeated 3 times by stirring with TBST for 10 min. After adding the ECL solution, the band was confirmed and quantified using the ChemiDoc Imaging System.To evaluate the protein expression of tyrosinase, microphthalmia transcription factor (MITF), TRP-1, and TRP-2 factors (whitening expression factors), B16F10 cells were seeded in a 100 mm tissue culture dish at 1 × 10t-test (Student’s t-test) were performed to determine any differences between the treatment conditions.The mean and standard deviation of each measurement group were calculated using the Statistical Package for the Social Science program , and the difference between the means of the measured values was independent. A one-way ANOVA and sample This study analyzed the functional components, antioxidant activity, and skin-whitening effect of sub- and SCW-treated rutin. The temperature/pressure conditions tested were 200 °C/15 bar, 300 °C/100 bar, and 400 °C/250 bar. The highest quercetin content (381.39 mg/g) was observed in 200 °C/15 bar-treated rutin, which decreased as the temperature and pressure increased. Although the quercetin content was lower at 300 °C/100 bar, and 400 °C/250 bar than at 200 °C/15 bar, the ABTS and DPPH assay showed the highest values at 300 °C/100 bar. The tyrosinase inhibitory activity was also the highest at 300 °C/100 bar. After solvent fractionation, the ABTS and DPPH radical scavenging activities, reducing power, and tyrosinase inhibitory activity were the highest in the ethyl acetate fraction. The ethyl acetate fraction showed 14.91% inhibitory activity on melanin synthesis at a concentration of 10 µg/mL. The inhibition rates of MITF, tyrosinase, TRP-1, and TRP-2 in the ethyl acetate fractions were 14.05%, 72%, 93.05%, and 53.44%, respectively, at a concentration of 10 µg/mL. In summary, when rutin is treated with SCW, various reactions that can affect the structure of components, including decomposition reactions, occur. As a result, rutin is decomposed or synthesized to create new substances, which can cause changes in various physiological activities. Therefore, SCW treatment can be utilized to develop cosmetic materials and functional food with enhanced physiological activity. The results of this study also indicated that SCW-treated rutin can be used as a skin-whitening cosmetic material. Future studies are needed to investigate the mechanisms for increasing the antioxidant and skin-whitening activity of SCW-treated rutin and identify the active compounds.


In this work, a kind of biphenyl-based dihydroxy monomer is synthesized using 4, 4'-biphenyl (BP) and triethylene glycol (TEG) as raw material, which further reacts with three different dicarboxylic acids  by melt polycondensation to prepare intrinsically highly thermally conductive poly 4', 4”'--bis(p-hydroxybiphenyl) succinate (PEOS), poly 4', 4”'--bis(p-hydroxybiphenyl) p-phenyldiacetate (PEOP) and poly 4', 4”'--bis(p-hydroxybiphenyl) terephthalate (PEOT), collectively called biphenyl-based liquid crystal polyesters (B-LCPE). The results show that B-LCPE possess the desired molecular structure, exhibit smectic phase in liquid crystal range and semicrystalline polymers at room temperature, and possess excellent intrinsic thermal conductivities, thermal stabilities, and mechanical properties. λ of PEOT is 0.51 W/(m·K), significantly exceeds that of polyethylene terephthalate (0.15 W/(m·K)) which has similar molecular structure with PEOT, and also higher than that of PEOS (0.32 W/(m·K)) and PEOP (0.38 W/(m·K)). The corresponding heat resistance index (THRI), elasticity modulus, and hardness of PEOT are 174.6°C, 3.6 GPa, and 154.5 MPa, respectively, and also higher than those of PEOS  and PEOP .Enhancing thermal conductivity coefficient ( The exp1H NMR and 13C NMR spectra of intermediate compound (TGT) and dihydroxy monomer (EOEH), and the FT-IR spectra of TGT and EOEH are shown in Figure −1 does not appear in the spectrum of TGT, while there are abundant absorption peaks appearing at 700~900 cm−1 (illustrating the presence of benzene rings in the structure of TGT). The disappearance of hydroxyl groups and the appearance of benzene rings further confirm the successful synthesis of TGT. In addition, compared with BP, EOEH shows stretching vibration peaks of -CH2- near 2900 cm−1 and absorption peaks attributed to C-O-C at 1000~1300 cm−1, further proving that the molecular structure of EOEH is consistent with expected design.Figure 1H NMR, 13C NMR, and FT-IR spectra of B-LCPE and presents the corresponding molecular weight and molecular weight distribution of B-LCPE. From 1H NMR spectra  are from the out-of-plane bending vibration of C-H in benzene rings, which further confirms the existence of ester groups and indicates the success of melt polycondensation. Results of GPC in Figures n) of PEOS, PEOP, and PEOT are 16.2 kg/mol, 16.3 kg/mol, and 16.1 kg/mol, respectively, all of which are very close to each other and reach prescriptive order of magnitudes for polymers. And polydispersity indexes (PDI) are all less than 1.2, illustrating that B-LCPE have narrow molecular weight distribution, basis of good molding quality, and excellent performances. Figures , the cha Figures  belong tn Figure , indicatT1 of crystal) during heating process corresponds to the transition from crystalline state to liquid crystal state, while the second endothermic peak (the peak temperature is the clearing point T2) corresponds to the transition from liquid crystal state to isotropic liquid state. T1 for PEOS, PEOP, and PEOT are 93.0°C, 94.3°C and 80.2°C, and the corresponding T2 are 183.8°C, 185.5°C, and 182.3°C, respectively. Moreover, the DSC results of cooling process further indicate that all B-LCPE are bidirectional thermotropic liquid crystal polymers. As temperature decreases, the first exothermic peak (the peak temperature is denoted by T3) corresponds to the transition from isotropic liquid state to liquid crystal state, and the second exothermic peak  corresponds to the transition from liquid crystal state to crystalline state. T3 of PEOS, PEOP, and PEOT are 140.7°C, 152.2°C, and 135.0°C, respectively, and T4 in the same order are 57.2°C, 55.0°C, and 72.0°C. It is worth noting that under the same nonisothermal crystallization condition, T4 of PEOT is significantly higher than that of PEOS and PEOP, proving that PEOT has a relatively strong crystallization ability, which is conducive to the formation of larger-sized crystalline grains. Otherwise, the DSC results of cooling process show great degree of subcooling, which is the normal phenomenon for polymers with viscosity , where T5 and T30 are decomposition temperatures with 5% and 30% mass loss, respectively [5 + 0.6∗T − T5], wπ-π stacking interaction increases intermolecular forces and makes PEOT arrange regularly and compactly at molecular scale, and possess the highest crystallinity, thus endowing PEOT superior mechanical properties.λ of PEOT is 0.51 W/(m·K), significantly exceeds that of polyethylene terephthalate (0.15 W/(m·K)), and is higher than that of PEOS (0.32 W/(m·K)) and PEOP (0.38 W/(m·K)). The corresponding heat resistance index (THRI), elasticity modulus and hardness of PEOT are 174.6°C, 3.6 GPa, and 154.5 MPa, respectively, and also are higher than those of PEOS  and PEOP .B-LCPE possess the desired molecular structure, exhibit smectic phase in liquid crystal range and semicrystalline polymers at room temperature, and possess excellent intrinsic thermal conductivities, thermal stabilities, and mechanical properties. 2CO3 solution and distilled water for three times, respectively. The organic phase was dried using anhydrous Na2SO4. DCM was then evaporated with rotary evaporator to obtain yellow liquid. Finally, recrystallization was conducted with ethyl alcohol (EtOH) as solvent, followed by drying in 60°C vacuum oven to obtain TGT (yield: 95.0%).6.3 g of 4-toluene sulfonyl chloride (TsCl) was dissolved in 35.0 mL of dichloromethane (DCM), and 2.0 mL of triethylene glycol (TEG) and 4.6 mL of triethylamine (TEA) were added drop-wise under stirring in ice-water bath. After reacting for 12 hrs, the reaction mixture was washed by saturated Na2CO3 and 3.5 g of KI were added into the above solution, and the mixture reacted at 80°C for 48 hrs. Afterwards, the hot reaction mixture was filtered, while the residue was washed by THF. After repeating for three times, THF in the collected filtrate was removed by rotary evaporator. Then, the residue was washed successively by saturated Na2CO3 solution, distilled water, EtOH, and DCM, followed by drying in 60°C vacuum oven to obtain EOEH as white solid, namely, biphenyl-based dihydroxyl monomer (yield: 56.0%).6.8 g of TGT and 5.6 g of 4, 4'-biphenyl (BP) were dissolved in 50.0 mL of tetrahydrofuran (THF) followed by magnetically stirring. 20.7 g of K2) protection before reaction. Afterwards, 0.3 wt% Sb2O3 and 0.2 wt% ZnAc2 were added into the flask as catalyst. Then, the flask was heated to 160°C in N2 atmosphere. When the mixture was homogeneous, the reaction was kept for 2 hrs under magnetic stirring. Meanwhile, the flow rate of N2 was increased to blow away by-product. Then, the reaction mixture was heated to 180°C (heating rate was 0.33°C/min) and reacted for 2 hrs. Afterwards, the temperature rose to 200°C and kept for 3 hrs under high vacuum. After that, the reaction mixture was allowed to cool to room temperature under vacuum to obtain tan solid, which was then dried at 60°C under vacuum for overnight (yield: 67.9%). Then, the synthesized B-LCPE were molded by casting method: The solids were added into the mold and heated to 220°C for melting about 20 min and then cooled to room temperature for demolding.B-LCPE were prepared following the same procedure. Typical synthetic procedure of PEOS was as follows: 14.6 g of EOEH and 3.5 g of succinic acid were loaded into reaction flask equipped with mechanical stirrer. To remove oxygen, the flask was processed by vacuum pumping and nitrogen (NMain materials and Characterizations are detailed in Supplementary Materials.


Cell surface expression of sortilin in different types of cancer signifies it as a therapeutic target for cancer therapy. The aim of this study was to detect sortilin expression in bladder cancer cells using an anti-sortilin monoclonal antibody (mAb) to evaluate sortilin as a target for developing diagnostic and therapeutic agents against bladder carcinoma. The protein expression of sortilin in bladder cancer tissues and cell lines (5637 and EJ138) was investigated by immunohistochemistry (IHC), immune-cytochemistry (ICC), and flow cytometry. Furthermore, the capability of anti-sortilin mAb in apoptosis induction in bladder cancer cells was evaluated.P≤0.001) and cell lines, using IHC and ICC, respectively. Flow cytometry results showed cell surface expression of 27.5±3% (P≤0.01), 74.4±7.8% (P≤0.001), and 4.2±0.4% of sortilin in EJ138, 5637, and HFFF cells, respectively. In EJ138 anti-sortilin mAb induced apoptosis in 25.2±11.5% (P≤0.05) (early) and 4.5±1.1% (P>0.05) (late) after 6 h incubation, while for 12 h, the values of 11.6±3.8% (P>0.05) and 20.7±4.4% (P≤0.05) were achieved. In 5637 cells, 6 h incubation resulted in 10.2±0.3% (P>0.05) and 6.6±1.4% (P>0.05) apoptosis induction, while these values were 12.1±0.8% (P>0.05) and 27.4±4.5% (P≤0.01) after 12 h. The HFFF cells did not show significant apoptosis. A high expression level was observed in bladder carcinoma tissues (The overexpression of sortilin in bladder tumor cells and its potential in inducing apoptosis via directed targeting with the specific monoclonal antibody may represent this protein as a potential candidate of targeted therapy in bladder carcinoma. Cancer is the second cause of human death world-wide, with more than 9.5 million cases per year. Among those, about 200.000 cases are bladder cancers. Bladder cancer is the fourth most common malignant with most male patients . This maSORT1 gene located on the short arm of chromosome 1 (1p13-3). Structur-ally, sortilin is a type I transmembrane glycoprotein receptor with an extracellular domain, a single transm-embrane helix, and a short cytoplasmic tail , is a multi-ligand receptor and a member of the Vacuolar Protein Sorting 10 (VPS10) family of sorting receptors that is involved in various biological processes -15. Sortmic tail , 17. The overexpression and dysregulation of sortilin in several human malignancies have been reported previo-usly, representing sortilin as a cell surface protein appro-priate for targeted immunotherapy using a monoclonal antibody -20. AlthIn our previous study, we produced a monoclonal antibody called 2D8–E3 against a synthetic peptide derived from the first 50 amino acids of the extrace-llular domain of sortilin, capable of recognizing its corresponding protein . This stCell Culture2 and 95% humidity , penicillin, and streptomycin were purchased from Gibco, NY, USA. Human bladder carcinoma cells lines EJ138 , 5637 , and human Caucasian fetal foreskin fibroblast  cells were obtained from National Cell Bank of Iran . All cell lines were cultured in RPMI-1640 containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL) and incubated at 37°C with 5% COhumidity .Immunohistochemistry (IHC) 2O2 was used. To block the nonspecific binding sites, a 5% normal sheep serum in Tris-buffered saline containing 2.5% Bovine serum albumin (2.5% TBS-BSA) was used. The slides were then incubated with 10 µg/mL anti-sortilin mAb  in a 2.5% TBS-BSA or anti-beta actin or mouse IgG isotype control antibodies  at room temperature (RT) for 60 min. The slides were washed by 0.1% TBS-BSA for three times, and 50 µL of EnVision reagent  was added to the slides with an incubation time of 30 min at RT. Then, 50 µL of DAB substrate    was added to each slide with subsequent adding hematoxylin dye . Finally, the slides were washed with deionized water, followed by dehydrating with ethanol, mounting by Entellan . The slides were examined using fluorescent microscopy . Formalin-fixed paraffin-embedded (FFPE) from human bladder carcinoma (n=23) and normal bladder tissue samples (n=20) were received from Imam Khomeini hospital, Tehran, Iran, and National Forensic Organization, Tehran, Iran, respectively. Tissues were deparaffinized and prepared for immunostaining accor-ding to our previous report . To quenImmunocytochemistry (ICC)4 cells/well in 100 µL medium on an eight wells glass coverslip  and incubated overnight with complete RPMI-1640  at 37°C in 5% CO2 with humidity atmosphere. Then cells were fixed by acetone for 10 min, blocked with 5% normal sheep serum for 30 min, and incubated with 5 µg/mL sortilin mAb as well as IgG isotype control for 45 min at RT. The slides were further incubated with secondary FITC-conjugated sheep anti-mouse Ig  at a dilution of 1:50 for an additional 30 min. The nuclei were stained with 1 µg/mL 4`,6-diamidino-2-Phenylindole (DAPI)  stain for 5 min. The slides were examined under a fluorescent microscope   .Flow CytometryAll three cell lines were cultured to reach 70–80% confluency. The cells were harvested and washed using pre-cold phosphate-buffered saline (PBS) and blocked with 5% sheep serum at 4°C for 30 min. The cells were then treated for 1 h at 4°C by 10 µg/mL concentration of anti-sortilin mAb or equivalent concentration of IgG isotype control antibody. The cells were washed as described above, incubated with FITC-conjugated sheep anti-mouse for 45 min at 4°C (in a dark place), at 1:50 dilution. The washing was repeated, and the fluorescence was measured using a Partec PAS® flow cytometer . The results were analyzed with FloMax software . To determine the relative total cell surface expression of sortilin, the mean fluore-scence intensity (MFI) was multiplied by the perce-ntage of positivity (POP) (MFI×POP) . Apoptosis Assay5 cells from each cell line were cultured in a 24 well plate in RPMI- 1640 medium. The procedure was followed by treatment with 10 µg/mL of 2D8-E3 mAb or an isotype control IgG for 6 and 12 h in a serum-free medium. The cells were harvested and washed twice with cold PBS. Finally, 100 µL of the cell suspension in 1x binding buffer was stained with 1 µL Annexin V- FITC  and 2 µL propidium iodide (PI) , followed by a short vortex and incubation at RT for 15 min in a dark place. The percentage of early apoptotic cells  and late apoptotic cells , as well as live cells , were observed using Partec PAS III flow cytometer  and analyzed by FlowJo software (version 10)  . Statistical Analysis .The statistical analysis was carried out by one-way and two-way ANOVA using GraphPad Prism software. The results were illustrated as mean ± SD. The P-values less than 0.05 were considered statistically significantImmunohistochemical Staining for Evaluation of Sortilin ExpressionP≤0.001)  . Detection of Sortilin by Immunocytochemistry The expression of sortilin in bladder cancer cells was also investigated by immunofluorescent staining using anti-sortilin mAb (2D8-E3). Both EJ138 and 5637 cells showed expression of sortilin (the green color) detected by their interaction with 2D8-E3, while no signal was detected in the normal cell line (HFFF) . Cell Surface Sortilin Detection by Flow Cytometry P≤0.01) and 5637  bladder carcinoma cell lines. In comparison, HFFF normal control cell line expressed low amount of sortilin (count: 4.2±0.4%). The arbitrary values of MFI×POP for EJ138, 5637, and HFFF cells were 610.5, 2641.2, and 10.24, respectively  (early) and 4.5±1.1% (P>0.05) (late) apoptosis was detected, while for 12 h incubation, the values of 11.6±3.8% (P>0.05) (early) and 20.7±4.4% (P≤0.05) (late) were achieved in EJ138 (P>0.05) (early) and 6.6±1.4% (P>0.05) (late) apoptosis induction, while these values were 12.1±0.8% (P>0.05) (early) and 27.4±4.5% (P≤0.01) (late) after 12 h incubation. The HFFF cells almost did not show significant apoptosis. Only 2.1±1.5% (early) and 0.2±0.1% (late) of apoptotic cells were detected following 6 h incubation. These values were 0.3±0.2% (early) and 2.8±0.3% (late) after 12 h incubation.EJ138, 5637, and HFFF cells were treated with anti-sortilin mAb for 6 and 12 h. The results indicated apoptosis induction in both carcinoma cell lines. After 6 h incubation, 25.2±11.5%  is more than the normal fetal foreskin fibroblast cell line (HFFF);. However, the expression in the 5637 cell line is higher than EJ138. This finding may be due to the chara-cteristics of each cell line representing its particular phenotype and duty. Phenotypically, EJ138 is a transitional bladder cell carcinoma and known as an invasive cell line, while the 5637 is a grade II bladder carcinoma and known to be a non-invasive line , 32. FurIn this study, we also evaluated the functional role of sortilin in the pathophysiology of bladder carcin-oma, using apoptosis induction assay with anti-sortilin mAb in cell culture. The results showed that anti-sortilin mAb induced apoptosis in both EJ138 and 5637 cell lines after 6 and 12 h treatment without significant effect on human normal HFFF cells. Interestingly, the early apoptosis rate was similar in both cells after 6 h, while the percentage of late apoptosis was higher in 5637 cells than EJ138 cells. After 12 h, the early and late apoptosis rate was elevated in 5637 cells compared to EJ138 cells. However, no apoptosis was detected in comparison with an isotype control. The observation of apoptosis in EJ138 after 6 h might reflect the role of sortilin as a survival factor in bladder cancer cells. The notion has previously been described for other cancer cell types like neuroendocrine tumors and B cell malignancies , 40. SevSome reports demonstrated that sortilin is a contributory factor in expanding cancer stem cells. This is due to the role of sortilin in the internalization of progranulin as a glycoprotein involved in the transforming machinery and propagation of cancer stem cells , 47. In The overexpression of sortilin in bladder tumor cells and its potential in inducing apoptosis via directed targeting with the specific monoclonal antibody may represent potent as a potential candidate of targeted therapy in bladder carcinoma.The authors declare no conflict of interest.None.


Accumulating evidence shows that oxidative stress and inflammation contribute to the development of cardiovascular disease. It has been suggested that propolis possesses antioxidant and anti-inflammatory activities. In this study, the antioxidant and anti-inflammatory effects of the main flavonoids of propolis  and propolis extract were researched. The results showed that the cellular ROS (Reactive oxygen species) levels, antioxidant enzymes, Nrf2 (Nuclear factor erythroid 2-related factor 2) nuclear translocation, and the expression of NQO1 (NAD(P)H:quinone oxidoreductase 1) and HO-1 (heme oxygenase 1) were regulated by different concentrations of individual flavonoids and propolis extract, which showed good antioxidant and pro-oxidant effects. For example, ROS levels were decreased; SOD and CAT activities were increased; and the expression of HO-1 protein was increased by chrysin. The results demonstrated that NO (Nitric Oxide), NOS (Nitric Oxide Synthase), and the activation of the NF-κB signaling pathway were inhibited in a dose-dependent manner by different concentrations of individual flavonoids and propolis extract. Moreover, the results revealed that the phytochemicals presented antioxidant effects at lower concentrations but pro-oxidant effects and stronger anti-inflammatory effects at higher concentrations. To maintain the balance of antioxidant and anti-inflammatory effects, it is possible that phytochemicals activate the Nrf2 pathway and inhibited the NF-κB (Nuclear factor kappa B) pathway. Cardiovascular disorders such as myocardial infarction are considered to be among the leading causes of mortality. Cardiovascular disorders produce excessive oxygen free radicals in the pathological process, and the disorders of free-radical metabolism in the state of oxidative stress are important triggers of myocardial damage . One of Numerous research studies confirmed that oxidative stress and inflammation are interdependent and interrelated. The pro-inflammatory response could be enhanced by oxidative stress. For instance, it was demonstrated that the inflammatory phenotype of mice was modified via the genetic regulation of antioxidant defenses and ROS-generating enzymes ,5. In adAdditionally, oxidative stress caused by ROS was recently regarded as a possible mechanism in cardiovascular diseases. Numerous genes associated with oxidative stress are regulated by critical transcription factor Nrf2 . Upon acIn recent years, studies showed that propolis has antioxidant, anti-inflammatory, anti-cancer, anti-bacterial, and hepatoprotective properties. Propolis is widely used as a functional food to promote public health and prevent chronic conditions such as atherosclerosis ,17, typeTherefore, the fundamental mechanisms of antioxidant and anti-inflammatory activities in H9c2 cells were investigated using propolis extract and its main flavonoids at different concentrations. H9c2 cells are usually used to simulate heart disorders, myocardial diseases, cardiovascular diseases, etc. For example, H9c2 cells were used to induce oxidative stress in order to simulate septic cardiomyopathy by Jiang et al. ; to indu2O2) were bought from Damao Co. Ltd. . Cell Counting Kit-8, Total Superoxide Dismutase Assay Kit with WST-8, Catalase Assay Kit, Griess Reagent, Nitric Oxide Synthase Assay Kit, Dichlorodihydrofluorescein-Diacetate (DCF-DA), sodium dodecyl sulfate (SDS) loading buffer (6×), sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-enhanced chemiluminescence detection reagents were bought from Beyotime Biotechnology . Polyvinylidene fluoride (PVDF) membranes were bought from Roche Diagnostics GmbH . VCAM (1:3000), Nrf2 (1:1000), HO-1 (1:3000), and NQO1 (1:3000) were purchased from Abcam . Phospho-NF-κB p65 (1:1000), IL-6 (1:1000), and Histone H3 (1:3000) were bought from Cell Signaling Technology . β-actin (1:3000) was bought from Santa Cruz . Horseradish-peroxidase-conjugated anti-rabbit (1:3000) and anti-mouse (1:3000) were bought from Signalway Antibody .Propolis was bought from Jiangxi Nanchang Tongxinzicao Biological Engineering Co. Ltd.  and had been gathered in September 2015 in Nanchang . H9c2 cells were bought from Procell Life Science&Technology Co., Ltd. ; it is a clonal cell line subclonally obtained from BD1X rat embryonic heart tissue; Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were bought from Biological Industries . Penicillin–streptomycin liquid, Tris-buffered saline–Tween-20 (TBST), pinobanksin standards, and pinocembrin standards were bought from Solarbio Life Sciences . Acetone, methanol, dimethyl sulfoxide (DMSO), and 30% hydrogen peroxide (Hw/v) in ethanol–water solution  and ultrasonicated  at the power of 100 W and 20 kHz. The mixture was centrifuged  at 4200× g for 5 min. Subsequently, the supernatant was gathered, and the extraction of the residue was repeated four times at least under the same conditions as before. After that, the supernatants obtained in multiple extraction experiments were mixed together, condensed under vacuum at 37 °C, and finally lyophilized for further study. The preparation of propolis extract was performed as follows: Propolis was de-mixed and frozen at −18 °C for 24 h; then, it was crushed with a pulverizer and sieved through a 120-mesh sieve, and the finished powder was stored at −80 °C. Propolis powder was immersed in a ratio of 1:30 . Chromatographic separation was implemented on an Agilent Eclipse XDB C18  column with detection being carried out at 280 nm with the operating temperatures being kept at 35 °C. The analyses were completed with a gradient elution of methol (A) and 0.1% formic acid in purified water (B). The gradient protocol was: 22–36% A, 0–5 min; 36–52% A, 5–30 min; 52–63% A, 30–65 min; 63–70% A, 65–95 min; 70–80% A, 95–120 min; 80–22% A, 120–122 min. The injected sample volume was 5 μL, and the flow rate was 0.6 mL/min.m/z = 50–2000.An Agilent 1260 HPLC (quaternary pump) system and AB Sciex TripleTOF™ 5600 mass spectrometer were used in the ESI-QTOF-MS/MS system. An electrospray ionization source was used to drive the TOF mass spectrometer. The capillary voltage was set to 4 kV; the collision voltage was set to 135 V; the drying-gas temperature was set to 350 °C; the drying-gas flow rate was set to 10 L/min; the nebulizer pressure was set to 40 psi; the collision gas was nitrogen; the collision energy was set to 30 eV; the full ionic scan mode was used; and the scan range was set to 2. Cells were seeded into appropriate dishes or plates for 24 h before being subjected to various treatments. Chrysin, pinobanksin, galagin, and pinocembrin were dissolved in DMSO as reserve solutions (10 mM). All reserve solutions were stored at −80 °C and diluted into DMEM at various ratios prior to cellular incubating.H9c2 cells were plated into DMEM containing inactivated 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were incubated in an incubator at 37 °C with 5% CO5 cells per well) were seeded in a 96-well plate and allowed to complete from 80% to 90% confluence before being treated. Subsequently, the cells were exposed to various different treatments. Then, CCK-8 reagent (10 μL) was combined with 90 μL of DMEM to generate a working solution. After that, 100 μL of CCK-8 working solution was transferred towards each well, and the cells were additionally cultured for 1 h. Ultimately, the absorption at 450 nm was monitored with a microplate reader.H9c2 cells (1 × 106 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. Cells were treated with standards and propolis extract for 12 h, followed by inducing with 150 μM H2O2 for 1 h, and then incubated with 5 mM DCF-DA (fluorescent probe) in DMEM at 37 °C for 15–20 min in the dark. Cells were digested with 0.02% EDTA (ratios of commonly used cell lysis solutions that are recommended in flow cytometry) and then centrifuged at 1500 rpm for 10 min to remove the supernatant. The pellets were re-suspended in 200 μL of cold PBS. Fluorescence intensity was monitored via flow cytometry .H9c2 cells (1 × 106 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. After various treatments, upon lysis with a tissue lysis solution, the supernatant of H9c2 cells was collected to measure the SOD and CAT activities. The supernatant was then added to the corresponding 96-well plate in a volume of 50 μL. The intracellular SOD and CAT activities were determined according to the procedures of Total Superoxide Dismutase Assay Kit and Catalase Assay Kit. Finally, the absorbance at 560 nm (SOD) or 520 nm (CAT) was measured with a microplate reader . The supernatant was used to determine the protein concentration in the BCA protein-concentration assay kit. H9c2 cells (1 × 106 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. After various treatments, Griess Reagent was used to detect the release of NO. Then, we took a 96-well plate and placed 50 μL of cell culture medium into the corresponding wells. A total of 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to the corresponding wells. Nitric Oxide Synthase Assay Kit was allowed to equilibrate at 25 °C for 20 min. The treated 96-well plate was aspirated out of the culture solution, and 100 µL of NOS Assay Buffer was added. Another 100 µL of Assay Reaction Solution was added and gently mixed. Finally, the absorbance at 540 nm was measured with a microplate reader .H9c2 cells (1 × 106 cells per well) were cultured in culture dishes 3 cm in diameter and were allowed to reach from 80% to 90% of confluence before treatment. After various treatments, cells were cleaned three times with cold PBS. Whole-cell proteins were extracted using a protein extraction kit . Briefly, after adding 200 µL of lysate/dish, the culture dishes were placed in an ice-box for 30 min and then centrifuged at 12,000× g for 10 min; finally, the supernatant was obtained as whole-cell proteins. H9c2 cells  were mixed with sodium dodecyl sulfate (SDS) loading buffer , and proteins were detected via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes using constant current. After being blocked with 5% skimmed milk in TRIS-buffered saline–Tween-20 for 2 h at room temperature (about 25 °C), PVDF membranes were incubated with the corresponding primary antibodies VCAM (1:3000), Nrf2 (1:1000), HO-1 (1:3000), NQO1 (1:3000), phospho-NF-κB p65 (1:1000), IL-6 (1:1000), and Histone H3 (1:3000) or β-actin (1:5000) overnight at 4 °C. The membranes were washed with TBST and then incubated with horseradish-peroxidase-conjugated anti-mouse (1:3000) or anti-rabbit (1:3000) secondary antibodies in TBST for 2 h at room temperature. Finally, target bands were observed in enhanced chemiluminescence (ECL) detection solution using the ECL technique  in the dark after three washes with TBST. The intensity of the target bands was determined using an imaging analyzer . The relative expression of proteins was standardized to Histone H3 or β-actin. p < 0.05 was used to denote significant differences. The data were statistically analyzed using GraphPad Prism 8 software .The SPSS 26.0 statistical software tool  was used to analyze the data, which were given as mean standard errors of the mean (SEMs). Duncan’s tests were used to assess differences after a single-factor analysis of variance (ANOVA), and −) of Peak 8, the characteristic fragment ion ([M-H-CO2-CO]− = 211.0319) of Peak 16, and the characteristic fragment ion ([M-H-CH3-CO]− = 226.0548) of Peak 17 were identified as methoxyl-cyanidenon [− and 253.0419 [M-acetate-H2O]−), which may be related to the cleavage of acyl bonds . Peaks 12 and 18 were identified as 3-O-acetyl-pinobanksin (m/z = 313.0612) and 3-O-butyryl-pinobanksin (m/z = 341.0917), respectively, based on fragment ions, molecular ions, and the literature [Eleven flavonoids, including chrysin, galangin, pinocembrin, pinobanksin, pinobanksin 3-O-acetate, pinobanksin-3-O-butyric, pinobanksin-5-methyl ether, apigenin-7-O-glucoside, methoxyl-cyanide, galangin-5-methyl ether, and pinocembrin-7-methyl ether, were identified in propolis extract by comparing the coupling of HPLC-ESI-QTOF-MS/MS fragmentation with reference standards and literature information ,33,34,35anidenon . Peaks 1terature . From thterature , the mai2O2 concentrations  for 1 h. Compared with the control group, cell viability was 60.24% . The concentration with the highest intracellular ROS level was used in subsequent experiments. Cells were in a state of oxidative stress when the viability was 50–70%, which does not immediately cause death and can be recovered using antioxidants. Oxidation significantly harms cells and does irreparable damage when cell viability falls below 40%. Therefore, 150 μM H2O2 was used to induce cellular oxidative damage in H9c2 cells. To induce oxidative stress, H9c2 cells were exposed to multiple Hs 60.24%  when the 205.12%  when theThe viability of H9c2 cells was unaffected by LPS concentrations of 5, 10, 15, 20, 25, 30, 35, and 40 μg/mL . CompareThe CCK-8 assay was used to assess the potential cytotoxicity of chrysin, pinocembrin, galangin, pinobanksin, and propolis extract in H9c2 cells. When the concentration of chrysin was higher than 30 μΜ, that of galangin higher than 60 μM, and that of propolis extract higher than 100 μg/mL, cell viability was below 95%. However, pinocembrin below 300 μM and pinobanksin below 300 μM did not inhibit the proliferation of H9c2 cells . In orde2O2-induced ROS generation (p < 0.05), and significant differences were found among doses that showed a trend of first decreasing and then remaining constant or increasing. On the one hand, after chrysin, pinocembrin, and propolis-extract pretreatment, the antioxidant effects tended to first increase and subsequently decrease. On the other hand, after galangin and pinobanksin pretreatment, the antioxidant effects tended to first increase and subsequently stabilize.ROS are among the major intracellular oxidation products and important participants in cell signaling. Their accumulation probably causes macrophages to undergo more apoptosis or autophagy . Pretrea2O2-induced group (373.35 ± 2.42%), showed anti-oxidant effects in the concentration range from 5 μM (361.15 ± 1.57%) to 10 μM (243.38 ± 1.22%), while it showed pro-oxidant effects in the concentration range from 15 μM (331.01 ± 4.16%) to 25 μM (354.88 ± 1.58%). Pinocembrin (2O2-induced group (373.64 ± 17.37%), showed anti-oxidant effects in the concentration range from 5 μM (294.56 ± 7.6%) to 40 μM (179.94 ± 6.2%), while it showed pro-oxidant effects in the concentration range from 60 μM to 80 μM. Galangin (2O2-induced group (430 ± 1%), showed anti-oxidant effects in the concentration range from 10 μM (384.59 ± 0.87%) to 50 μM (232.27 ± 6.69%), while it showed pro-oxidant effects in the concentration range from 50 μM to 60 μM (240.7 ± 2.33%). Pinobanksin (2O2-induced group (461.88 ± 36.48%), showed anti-oxidant effects in the concentration range from 5 μM (320.97 ± 14.04%) to 40 μM (179.94 ± 6.2%), while it showed pro-oxidant effects in the concentration range from 40 μM to 80 μM (182.81 ± 4.3%). Propolis extract (2O2-induced group (331.87 ± 4.56%), showed anti-oxidant effects in the concentration range from 10 μg/mL (236.43 ± 3.16%) to 40 μg/mL (49.78 ± 2.6%), while it showed pro-oxidant effects in the concentration range from 60 μg/mL (112.79 ± 1.58%) to 100 μg/mL (223.64 ± 3.17%).Chrysin A, comparocembrin B, comparGalangin C, comparobanksin D, compar extract E, compar2O2-induced group (p < 0.05). Furthermore, after pretreatment with different sample concentrations, SOD and CAT levels were increased in H9c2 cells induced by H2O2 (p < 0.05). For example, as shown in 2O2 group , SOD activity (296.97 ± 2.23–298.56 ± 1.59 U/mg protein) and CAT activity (2.06 ± 0.11–3.04 ± 0.05 U/mg protein) were increased after 5–25 μM chrysin pretreatment.SOD plays an essential role in the conversion of superoxide to hydrogen peroxide, while CAT converts hydrogen peroxide to water . To expl2O2-induced H9c2 cells (p < 0.05). In addition, the expression of HO-1 and NQO-1 proteins was dramatically increased after pretreatment with different sample concentrations in H2O2-induced H9c2 cells  to protect the body from ROS damage ,42. Upon2O2-induced group (80.73 ± 0.88%), the expression of HO-1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 μM (98.85 ± 3.22%) to 15 μM (95.31 ± 1.02%), while the expression of HO-1 was gradually decreased and showed pro-oxidant effects in the concentration range from 20 μM (78.69 ± 9.44%) to 25 μM (62.59 ± 0.06%). On the other hand, after galangin and pinobanksin pretreatment, the expression of the HO-1 protein tended to first increase and subsequently stabilize. For example, with pinobanksin (2O2-induced group (46.7 ± 0.74%), the expression of HO-1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 μM (75.13 ± 0.83%) to 40 μM (96.85 ± 3.82%), while the expression of HO-1 was no longer consistently elevated and showed pro-oxidant effects in the concentration range from 40 μM to 80 μM (105.95 ± 4%).On the one hand, after pretreatment with chrysin, pinocembrin, and propolis extract, the expression of the HO-1 protein tended to first increase and subsequently decrease. For example, with chrysin A, comparobanksin D, compar2O2-induced group, there were no significant differences in the expression of NQO1 after chrysin pretreatments (p > 0.05). However, after galangin, pinobanksin, and propolis-extract pretreatments, the expression of the NQO1 protein tended to first increase and then stabilize. For example, with propolis extract (2O2-induced group (60.36 ± 0.38%), the expression of NQO1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 μM (90.88 ± 4.4%) to 40 μM (106.87 ± 7.5%), while the expression of the NQO1 protein was no longer consistently elevated and showed pro-oxidant effects in the concentration range from 40 μM to 100 μM (114.55 ± 1.97%). In addition, with pinocembrin (2O2-induced group (82.59 ± 0.84%), in the concentration range from 5 μM (90.50 ± 2.3%) to 80 μM (107.87 ± 0.37%), the expression of the NQO1 protein was significantly increased.Compared to the H extract E, comparocembrin B, compar2O2-induced group (p < 0.05). After treatment with different concentrations of chrysin  and then down-regulated  following treatments with chrysin, pinocembrin, and propolis extract. It was also found that Nrf2 translocation from the cytoplasm to the nucleus was up-regulated and then held relatively constant following treatments with 10–60 μM galangin and 5–80 μM pinobanksin.p < 0.05; NO is catalyzed by NOS in cells, and it can trigger tissue damage and ultimately lead to pain and inflammation ,45. The p < 0.05; In addition, NOS generates NO, which is an inflammatory mediator . The levp < 0.05; 167.29–245.79%) was significantly up-regulated in the LPS-induced group. After pretreatment with the samples, the expression of IL-6 and VCAM proteins was significantly inhibited in LPS-induced H9c2 cells in a dose-dependent manner  and VACM proteins  have a stronger oxidative activity, while flavonoids have a lower oxidative activity [2=C3 double bond plays an important role in the antioxidant activity of flavonoids. Chrysin and galangin both feature C2=C3 double bonds, but galangin differs in that it has a C-3 hydroxyl group that promotes the oxidation to benzoquinone intermediates. However, the oxidation of galangin requires the consumption of free radicals in the system. Due to the lack of a B-ring ortho-hydroxyl group, the pro-oxidation effect of galangin is weaker than that of chrysin [Firstly, the structure of a flavonoid indicates whether it has pro- or antioxidant effects. The structures of chrysin, pinocembrin, galangin, and pinobanksin are shown in activity ,49. It i chrysin . TherefoSecondly, chrysin, pinocembrin, galangin, pinobanksin, and propolis extract exhibited antioxidant and pro-oxidant effects in a dose-dependent manner. The occurrence of pro-oxidant effects may be due to the auto-oxidation of flavonoid compounds, because as the concentration of these flavonoids increases, so does the synthesis of lipid peroxidation products and the development of superoxide anion radicals . MoreoveThe individual flavonoids  and propolis extract exerted strong antioxidant effects as inducers of the Nrf2/HO-1 axis, and they were potent activators of Nrf2 nuclear translocation in H9c2 cells. The phytochemicals showed antioxidant effects at low concentrations and showed pro-oxidant effects at high concentrations, possibly by activating Nrf2. Once activated by oxidative stress, Nrf2 is translocated to the nucleus and binds to antioxidant transcription elements in the promoter region of phase 2 to increase the expression of certain antioxidant and detoxification genes, ultimately leading to cellular resistance to oxidative stress . HO-1 anOxidative stress and inflammatory responses are important components in the pathogenesis of cardiovascular stent disease. Accumulating evidence suggests that oxidative stress is inseparable from the inflammatory response. Currently, exploring the relationship between antioxidant and anti-inflammatory agents is a potential target for the prevention or mitigation of cardiovascular disease. Therefore, our study focused on the changes in the Nrf2 and NF-κB pathways involved in oxidative stress and inflammatory processes.According to the datas of ROS, NOS, and the expression of pro-inflammatory protein, chrysin showed anti-oxidant effects in the concentration range from 5 μM to 10 μM, while it showed pro-oxidant effects in the concentration range from 15 μM to 25 μM, and it showed anti-inflammatory effects in the concentration range from 5 μM to 25 μM. Pinocembrin showed anti-oxidant effects in the concentration range from 5 μM to 40 μM, while it showed pro-oxidant effects in the concentration range from 60 μM to 80 μM, and it showed anti-inflammatory effects in the concentration range from 5 μM to 80 μM. Galangin showed anti-oxidant effects in the concentration range from 10 μM to 50 μM, while it showed pro-oxidant effects in the concentration range from 50 μM to 60 μM, and it showed anti-inflammatory effects in the concentration range from 10 μM to 60 μM. Pinobanksin showed anti-oxidant effects in the concentration range from 5 μM to 60 μM, while it showed pro-oxidant effects in the concentration range from 60 μM to 80 μM, and it showed anti-inflammatory effects in the concentration range from 5 μM to 80 μM. Propolis extract showed anti-oxidant effects in the concentration range from 5 μg/mL to 40 μg/mL, while it showed pro-oxidant effects in the concentration range from 60 μg/mL to 100 μg/mL, and it showed anti-inflammatory effects in the concentration range from 5 μg/mL to 100 μg/mL. Additionally, the higher the concentration of a compound was, the stronger the anti-inflammatory effect was.The inhibition of the production of NOS induced by flavonoids is caused by their antioxidant properties, and these compounds can exert anti-inflammatory effects by scavenging ROS . FurtherNF-κB is a classical inflammatory signaling pathway that regulates the expression of immune genes encoding cytokines, such as IL-1β, TNF-α, and IL-6 . PreviouResearch demonstrates that a functional interaction and crosstalk between Nrf2 and NF-κB pathways exists to maintain balance or regulate oxidative stress and inflammation . Even thA total of 18 substances were identified in the ethanolic extract of propolis, including flavonoids and their derivatives, and phenolic acids and their esters. According to previous data, the main flavonoids of propolis extract were identical to chrysin (30.56 ± 0.60 mg/g DW), pinocembrin (30.96 ± 0.34 mg/g DW), galangin (16.69 ± 0.45 mg/g DW), and pinobanksin (9.36 ± 0.28 mg/g DW).The bioavailability values of galangin and chrysin in propolis extracts were determined in a study, and they were at 7.8% and 7.5%, respectively . MoreoveThe major flavonoids in propolis were identified as chrysin, pinocembrin, galangin, and pinobanksin. It was revealed that flavonoids from propolis mainly presented antioxidant effects at lower concentrations and presented pro-oxidant as well as stronger anti-inflammatory effects at higher concentrations. We observed anti-oxidant effects of chrysin in the concentration range from 5 μM to 10 μM, of pinocembrin in the concentration range from 5 μM to 40 μM, of galangin in the concentration range from 10 μM to 50 μM, of pinobanksin in the concentration range from 5 μM to 60 μM, and of propolis extract in the concentration range from 5 μg/mL to 40 μg/mL, while we observed pro-oxidant effects of chrysin in the concentration range from 15 μM to 25 μM, of pinocembrin in the concentration range from 60 μM to 80 μM, of galangin in the concentration range from 50 μM to 60 μM, of pinobanksin in the concentration range from 60 μM to 80 μM, and of propolis extract in the concentration range from 60 μg/mL to 100 μg/mL. In addition, we observed anti-inflammatory effects of chrysin in the concentration range from 5 μM to 25 μM, of pinocembrin in the concentration range from 5 μM to 80 μM, of galangin in the concentration range from 10 μM to 60 μM, of pinobanksin in the concentration range from 5 μM to 80 μM, and of propolis extract in the concentration range from 5 μg/mL to 100 μg/mL. Additionally, the higher the concentration of the compound was, the stronger the anti-inflammatory activity was. Flavonoids from propolis could probably activate the Nrf2 pathway and inhibit the NF-κB pathway to maintain the balance of antioxidant and anti-inflammatory effects. In the future, it is important to focus on the link between the Nrf2 and NF-κB pathways to explore the anti-oxidative mechanism of low-concentration flavonoids and the anti-inflammatory mechanism of high-concentration flavonoids.