Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide 3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA  close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a  As a consequence of the well documented and ever increasing prevalence of antibiotic resistant bacteria, new methods of combating resistant strains which are pathogenic are urgently required for both animal and human health.Escherichia coli carrying group 1 blaCTX-M, conferring resistance to third generation cephalosporin antibiotics. First described in 1986, the CTX-M β-lactamases, named for their high level of activity against cefotaxime (CTX), and first observed in Munich (M), are a group of rapidly spreading ESBLs and are now the dominant family, especially found in such bacteria as E. coli and K. pneumoniae  and polyamide (peptide) nucleic acids (PNAs) have proven successful in inhibiting the expression of a diverse range of targeted bacterial genes , demonstic entry . Howeveric entry .N-(2-aminoethyl) glycine units linked by peptide bonds  and a peptide conjugated PNA (P-PNA4), and a resultant phenotypic increase in sensitivity to the third generation cephalosporin antibiotic CTX in human clinical and veterinary field isolates.We have designed and developed two antisense oligo nucleotides, a 25-mer PMO (PMO1), and a 13-mer PNA (PNA4) to target areas of PMO1 was synthesized by GeneTools, LLC , with the base sequence of: agggtaccaattttttagtgacgcg (estimated purity > 92%). PNA4 was synthesized commercially by either Cellmano Biotech  (purity D 95.51%), or Cambridge Research Biochemicals  with the base sequence of: ggttattccttct. Peptide conjugated PMO1 (P-PMO1) and peptide conjugated PNA4 (P-PNA4) were synthesized commercially by Cambridge Research Biochemicals , with the amino acid sequence of the CPP of KFFKFFKFFK.Table 1. E. coli strains of human  origin were obtained from the Public Health laboratory, UK. AS19 was donated by Liam Good , BZ693/P, B3804 and LREC525 were from the collection of field strains at the Animal and Plant Health Agency. LREC90, used for control studies, carried a plasmid harboring blaCTX-M-14, which encoded β-lactamase from CTX-M-group 9. All other CTX resistant strains used in this study harbored variants of CTX-M-group 1.Strains used in this study are shown in blaCTX-M-15 cloned from a field isolate harboring plasmid pEK516 . This amplicon was TA cloned into cloning vector pCRTM2.1-TOPO (Invitrogen). It was screened on LB agar supplemented with CTX (2 mg/L) and cultured in liquid LB overnight. The recombinant plasmid was digested with BamHI and XbaI and the cloned insert isolated by gel purification. This was the ligated into the multi-cloning site (MCS) of the commercially available plasmid vector pET-9a (Novagen) and screened on LB agar supplemented with kanamycin (40 mg/L) and CTX (2 mg/L). The recombinant plasmid was designated pJBRCTX516. The successful construction of this plasmid was verified by restriction enzyme digestion and gel electrophoresis, and then by sequencing. An A to G point mutation at position +823 was corrected by site-directed mutagenesis using the GENEART® Site-Directed Mutagenesis System (Invitrogen).Plasmid pJBRCTX516 was constructed carrying a single copy of d pEK516 . ISEcp1 promoter . To ensu600 nm 0.6-0.8 (mid-log phase), with antibiotic supplements where required. Cell aggregates were disrupted by bead milling in a Retsch Mixer MM301 shaking homogeniser  at full speed for 5 min with 0.2-0.4 mm glass beads. The cells were collected by centrifugation at 10,000g for 2 min and the supernatant retained. A suitable volume of supernatant was incubated at 37° C with the desired concentration of CTX solution for varying amounts of time. Enzymatic activity was halted by transferring incubation solution (125 μl) to pre-prepared HPLC vials containing 125 μl phosphate buffer (pH2.2). The HPLC method used was adapted from t = 0 mins 18:82%, t = 5 mins 18:82%; t = 15 mins 45:55%; t = 16 mins 55:45%; t = 21 mins 55:45%; t = 22 mins 18: 82%; t = 30 next injection at a flow rate of 0.85 ml/min, with an injection volume of 25 μl per sample. CTX was detected in HPLC effluent at a wavelength of 254 nm. Analysis was performed with ChemStation software (Agilent Technologies). CTX was quantified using a standard calibration curve (0-400 mg/L CTX). The activity of blaCTX-M was expressed as CTX (μg) degraded per minute per bacterial cell.CTX-M enzyme activity was determined by measuring the degradation of CTX with an HPLC based assay. Overnight cultures of strains were prepared and grown in Mueller-Hinton Broth (MHB) to O.D.TM Mini Cell-Free Expression System (InvitrogenTM) was used for all in vitro protein expression studies. The manufacturer’s supplied protocol was followed and the reaction mixture was incubated with plasmid pJBRCTX516 (1 ng), purified by peqGOLD Plasmid Miniprep Kit (Peqlab® Ltd) and amount estimated by spectrophotometry. β-Lactamase activity was determined by HPLC as described above.The Expressway600 nm) was achieved. The culture was diluted to achieve a final cell density of approximately 100,000 CFU/ml, previously established empirically to be optimal for assessing the effect of antisense oligonucleotides, and for consistency across experiments. Diluted cell suspension (50 μl) was incubated with MHB (50 μl) growth medium supplemented with antibiotics and inhibitory agents where required, and transferred to a 96 well microtiter plate . CTX concentrations for blaCTX-M inhibition studies were set at a level required to increase the dormant lag phase by 1-3 h, as established in the current study (not shown). This was determined to be an optimal concentration to yield observable effects of synergistic antisense-CTX activity and was strain dependent. Bacterial cell growth was measured in a BMG Labtech FluoStar automated spectrophotometer  and optical density readings (600 nm filter) taken over 18-24 h. The O.D.600 nm filter readings were taken over 250 cycles, with 15 flashes per well per cycle approximately every 5 min. All replicates were independent cultures. Growth curves and statistical analyses were produced using Prism® 6 software (GraphPad). Minimum inhibitory concentrations (MICs) were determined to be the minimum antibiotic concentration observed by spectrophotometry required to completely inhibit growth in ≥50% of replicates over 18 h in the cell growth assay.The activity of antisense oligonucleotides was assessed by measuring their ability to restore the growth inhibitory effect of CTX in a cell growth assay. Bacterial cultures were typically grown from glycerol stocks at 37°C in a shaking incubator in MHB media containing CTX (2 mg/L) until early log phase  was cultured in the presence and absence of P-PNA4, P-PMO1, and CTX, and growth measured by spectrophotometry over 18–24 h. Minimum inhibitory concentrations (CTX) were established for LREC90 in the presence and absence of P-PNA4.Specificity and any inherent toxicity of peptide-conjugated and unmodified PNA4 and PMO1 were demonstrated in the following studies. A scrambled non-specific 25-mer PMO was incubated in the presence of pJBRCTX516 in a cell-free translation/transcription coupled system followed by the quantification of cefotaximase activity. AS19/pJBRCTX516 was cultured with the scrambled PMO in the presence and absence of CTX and growth measured by spectrophotometry over 18–24 h. Field isolates harboring blaCTX-M-15 mRNA . However, due to potential hairpin self-dimerization and homo-dimerization, the design was modified under the advice of the manufacturer (Gene Tools LLC) and the final design was complementary to mRNA positions -4 to +21, with a sequence of: agggtaccaattttttagtgacgcg . The target sequence was checked by NCBIs Basic Local Alignment Search Tool (BLAST) for homology with other bacterial and mammalian genes for potential unwanted off-target effects. This sequence was found by BLAST to be 100% complementary with all group 1 CTX-M β-lactamases. No bacterial off-target matches were found with greater than 72% sequence identity and no mammalian gene transcripts were found with a greater than 72% sequence identity.A 25 base PMO (PMO1) oligonucleotide was designed to target, by complementary base pairing, an area covering both the SD region and translational start codon region of blaCTX-M-15 mRNA  with 100% sequence complementarity to the extragenic region -17 to -5. This target sequence was checked by BLAST and found to be to be 100% complementary with all group 1 CTX-M β-lactamases. Four bacterial off-target matches were found with 92.3% sequence similarity (12 out of 13 bases). A large number (>50) of off-target mammalian transcripts were detected (13 out of 13 bases).A 13 base PNA (PNA4) was designed to target the SD region of Figure 2).Plasmid pJBRCTX516 was extracted and the successful recombination verified by restriction enzyme digestion and by sequencing (not shown). The plasmid vector contained T7 promoter and terminator regions flanking the MCS, making it a suitable vector for both cell-free and whole-cell inhibition experimental studies (P < 0.05) inhibited β-lactamase in a dose dependent manner (50-1000 nM). The following rates of CTX degradation were observed: uninhibited control sample: 1.01 μg/min/ng plasmid, 50 nM PMO: 0.34 μg/min/ng plasmid (66.05% inhibition), 100 nM PMO: 0.27 μg/min/ng plasmid (73% inhibition), 250 nM PMO: 0.15 μg/min/ng plasmid (85.33% inhibition), 500 nM PMO: 0.08 μg/min/ng plasmid (92% inhibition) and 1000 nM PMO: 0.04 μg/min/ng plasmid (96.08% inhibition). A non-complementary scrambled control PMO had no inhibitory effects on β-lactamase activity (not shown). PNA4, under the same conditions showed a similar, dose dependent effect ranging from 50% inhibition of CTX degradation at 50 nM PNA4, to 90% inhibition at 1000 nM PNA4.PMO1 significantly . When treated with CTX (2 mg/L), 20 μM PNA4 was sufficient to completely inhibit growth (not shown).An increased sensitivity to CTX (4 mg/L) was shown in a dose dependant manner (20-40 μM PMO1 and 5-30 μM PNA4), with growth completely inhibited at PMO1 concentration of 40 μM . In the absence of CTX, significant effects on growth were observed when AS19/pJBRCTX516 was challenged with PNA4 alone at concentrations higher than 5 μM . This was potentially attributable to unintended off-target partial complementarity with other bacterial genes. Consequently, PNA4 concentrations in subsequent studies did not exceed a concentration of 5 μM.There was no observed increase in CTX sensitivity when atypically permeable strain AS19/pJBRCTX516 was challenged with the combination of non-Field isolate LREC460 harboring the resistance plasmid pEK516 and field isolate LREC461 harboring the resistance plasmid pEK204, were incubated in Mueller Hinton Broth and challenged with CTX (16–32 mg/L) and PMO1/PNA4 (5–40 μM). It was found that neither PMO1 nor PNA4 had observable effects on growth or cell viability (data not shown). This was consistent with prior expectations as the cell wall has been previously shown to be a significant barrier to gene expression inhibition by antisense/antigene chemistries .E. coli isolates were selected and MICs to CTX established (Table 1). These strains were challenged with a range of P-PMO1 and P-PNA4 concentrations in combination with the concentration of CTX required to extend the lag phase by 1-3 h (Table 1).A panel of clinical and field blaCTX-M P-PMO1 (30 μM) ranging from a reduction in the MIC (to CTX) in blaCTX-M-3-harboring strain from 35 to 16 mg/L (2.19-fold increase in sensitivity), to a strain harboring blaCTX-M-1 from 48 to 10 mg/L (4.8-fold increase in sensitivity). The same isolates when treated with P-PNA4 (3.2 μM) had an observed increase in CTX sensitivity ranging from a reduction in MIC (to CTX) in blaCTX-M-3/33-harboring strain BZ693/P from 40 to 12 mg/L (3.3-fold) to a strain harboring blaCTX-M-15 (LREC454) from 110 to 2 mg/L (55-fold).P-PMO1 was tested in combination with CTX in a range of six field and clinical isolates harboring plasmids carrying different group 1 CTX-M genes. All isolates tested showed an increased sensitivity to CTX when treated with anti-blaCTX-M-15 were isolated by the treatment of cultures with P-PNA4 or P-PMO1 in the absence of CTX. In all strains tested, including strain LREC90 harboring blaCTX-M-14, both P-PMO1 and P-PNA4 in the absence of CTX individually had a small significant negative effect on growth, characterized by an extended lag phase period of variable, strain, and dose dependent amount. This was attributed to the effect of the CPP, which has been previously shown to have an impact on bacterial cell growth, and a synergistic effect with some antibiotics . No significant differences were observed between the effects on growth of LREC90 cultured in the presence of P-PNA4 alone (1.6–5 μM), and P-PNA4 (1.6–5 μM) co-administered with CTX (2 mg/L). Minimum inhibitory concentrations (CTX) of LREC90 were not reduced in the presence of P-PNA4 . A comparable effect on LREC90 (blaCTX-M-14) and LREC454 (blaCTX-M-15) growth was observed when cultured in the presence of P-PMO1 (10–30 μM), attributable to the peptide portion of the conjugate.Control studies, equivalent to the use of a scrambled non-specific P-PNA or P-PMO1, were undertaken in Figure 5).The negative effect on growth was increased in an additive manner  between 0.5 and 1) when LREC90 was cultured in the presence of P-PMO1 (10–30 μM) and CTX (3 mg/L), in contrast, the negative effect was increased in a synergistic manner (estimated FICI < 0.5) when LREC454 was cultured in the presence of P-PMO1 and CTX (24 mg/L)  and field isolate LREC460 (data not shown).The effect of exposure to an extended spectrum β-lactam on the activity of CTX-M-15 activity was investigated by pre-exposure to ceftriaxone  and subsequent quantification of CTX degradation. The rate of CTX degradation was increased in a dose dependent manner (2-64 mg/L) following exposure to higher levels of ceftriaxone in both AS19/pJBRCTX516  for phospholipid biosynthesis.The application of antisense oligonucleotides for the translational inhibition of specific genes, such as those that confer resistance to ESBLs, is an attractive potential strategy for the restoration of antimicrobial sensitivity. PMOs and PNAs have been previously successfully employed to inhibit bacterial protein expression in a targeted, sequence specific manner. A wide range of bacterial essential and non-essential gene transcripts have been targeted and inhibited by synthetic antisense oligonucleotides. For example, a 17-mer PNA was reported by blaCTX-M against a range of CTX-M group 1 β-lactamases in E. coli. This was demonstrated in a sequential series of progressive studies. β-lactamase was expressed in a cell free transcription/translation assay using a recombinant plasmid (pJBRCTX516) with a high amplitude promoter harboring blaCTX-M-15 as template DNA in the presence and absence of antisense oligonucleotides. β-lactamase activity was quantified by subsequent incubation with CTX, and degradation was quantified by an HPLC assay developed for the detection of CTX.In this study, we have designed and evaluated a 25-mer PMO (PMO1) and a 13-mer PNA (PNA4) antisense/antigene oligonucleotide for efficacy of inhibition of translation of E. coli strains with reduced susceptibility to CTX (Table 1) were ascertained by cell growth assays in the presence of CTX and antisense oligonucleotides.Increases in sensitivity to CTX in blaCTX-M-15 in a dose-dependent (50-1000 nM) manner. At 1000 nM, β-lactamase production was inhibited by 96.08%. A scrambled control PMO of the same size, 25 nucleotides, non-specific to any bacterial gene, was found to have no inhibitory effects on transcription/ translation at this genetic level. These data suggest a high sequence specific inhibitory potential of PMO1 against blaCTX-M-group1. Under the same conditions, PNA4 was found to inhibit production of blaCTX-M-15 in a dose-dependent (50–1000 nM) manner with a maximum inhibition at 1000 nM of 89.39%.In cell free studies, PMO1 was found to inhibit production of blaCTX-M-15. Specificity was demonstrated at the phenotypic level as a scrambled control PMO with no specificity to bacterial genes had no observable effects on cell growth. There were also no observable effects when AS19/pJBRCTX516 was incubated with the maximum concentration used in inhibition studies (40 μM) PMO1 in the absence of CTX. There was a small observable lag phase extension effect, in a dose dependent manner (2.5-20 μM), when AS19/pJBRCTX516 was incubated with PNA4. This was attributed to the off-target matches associated with the sequence design of PNA4.PMO1 was tested against the atypically permeable cell-wall mutant AS19, transformed with pJBRCTX516 harboring blaCTX-M-15. Under the same conditions, incubated in growth medium additionally supplemented with PNA4 (20 μM), the minimum inhibitory concentration was reduced to 2 mg/L. At 20 μM, PNA4 alone was insufficient to inhibit growth and had a synergistic relationship with CTX (FICI < 0.5).When incubated in the presence of both CTX and PMO1, increased antibiotic sensitivity was observed in a dose-dependent manner (10-40 nM). The MIC of CTX for this transformant, AS19/pJBRCTX516, was 80 mg/L, in the presence of 40 μM PMO1, this was reduced to 4 mg/L. These data demonstrated the lack of any inherent toxicity of PMO1 at the concentrations used, and the specific activity of PMO1 against E. coli field isolates LREC460 and LREC461 containing plasmids harboring CTX-M group 1 genes, neither PMO1 nor PNA4 was found to have any effect on their sensitivity to CTX, or to their growth characteristics. This was not unexpected as previous studies have shown that E. coli with an intact cell wall do not efficiently take up naked antisense oligonucleotides 3K, which has been shown to be effective in promoting uptake across the bacterial cell wall and delivering a cargo molecule to the cytoplasm (When tested with leotides . To overytoplasm . In the blaCTX-M target sequence (12 out of 13 bases), and an effect on growth was shown when challenging AS19/pJBRCTX516 with PNA4 alone. The off-target effects, however, were small in comparison with the synergistic effects of PNA4 in combination with CTX, and although significant (P < 0.05), were negligible at 3.2 μM. In the panel of six field and clinical isolates, P-PNA4 was found to be between 9- and 56-fold more effective in increasing sensitivity to CTX than P-PMO1.The disparity in the efficacy of P-PNA4 compared with P-PMO1 can, in part, be explained by the contribution of the off-target effects of the shorter PNA. Four bacterial gene transcripts shared 92.3% sequence identity with the 3K with the antibiotics gentamicin and amikacin. It is likely that at low concentrations, the mechanism of cell penetration of (KFF)3K compromises the cell wall and increases the uptake of antibiotics, as has been observed in other bacterial species  of the region targeted by P-PNA4, and 56% sequence complementarity (14 out of 25 bases) of the region targeted by P-PMO1. As such, this was regarded as a comparable control to scrambled non-specific antisense oligonucleotides in strains harboring blaCTX-M-15.Specificity and the effect on growth of the CPP were demonstrated at the phenotypic level by the exposure of A small effect on growth in the absence of CTX was observed, attributable to the CPP portion of the conjugate, potentially a result of membrane perturbation.The relatively modest increase in sensitivity to CTX of field isolates when co-administered with P-PMO1, contrasted with a much higher level of re-sensitisation in the cell wall compromised strain AS19 transformed with pJBRCTX516, and a near complete inhibition of protein expression when tested in a cell-free environment. This disparity suggested either inefficient uptake of the antisense agent, a higher level of efflux in field isolates, or greater β-lactamase expression in response to CTX challenge.We found experimental evidence to suggest that in conditions of increasing concentrations of CTX, in both field isolates and AS19/pJBRCTX516 there was a proportional up-regulation of β-lactamase activity. The differential expression mechanism requires further study.blaCTX-M in field isolates, and the potential for further optimisation and enhancement, demonstrated the potential for therapeutic use of P-PMOs and P-PNAs in re-sensitizing resistant bacteria to CTX. To our knowledge this is the first study using synthetic oligonucleotides to demonstrate inhibition of blaCTX-M group 1 genes in field and clinical isolates and restore sensitivity to the third generation cephalosporin CTX. Further studies are required to optimize uptake into the bacterial cell and translational inhibition.The data obtained from translational inhibition studies of JR: Ph.D. student, provided experimental work and wrote manauscript. GD: Academic PhD supervisor, provided guidance, and direction to experimental work and manuscript review/writing. NC: Industrial Ph.D. supervisor, provided guidance, and direction to experimental work and manuscript review/ writing.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.


The physiological and pathological relevance of this phenomenon and the underlying molecular mechanism, however, remain to be elucidated. B cells from wild type and EP2-deficient mice were compared in culture for their responses to PGE2 in terms of IgE class switching and production. Ovalbumin (OVA)-induced asthma models were used to evaluate the impact of EP2-deficiency on IgE responses and the development of asthma. PGE2 promoted IgE class switching, generation of IgE+ cells and secretion of IgE by B cells stimulated with LPS+IL4. These effects were much attenuated as a consequence of EP2 deficiency. Consistent with the in vitro data, EP2-deficient mice showed a markedly suppressed IgE antibody response and developed less pronounced airway inflammation in the OVA-induced asthma model. Mechanistic studies demonstrated that PGE2, in an EP2-depedent manner, enhanced STAT6 activation induced by IL-4, thereby promoting the expression of IgE germline and post switch transcripts and the transcription of activation-induced cytidine deaminase (AID). Collectively, these data support an important regulatory role of the PGE2-EP2-STAT6 signaling pathway in IgE response and allergic diseases.Prostaglandins E2 (PGE2) has been shown to enhance IgE production by B cells  Prostaglandins E2 (PGE2) is the most abundantly produced prostanoid in the body. Its production begins with the liberation of arachidonic acid from membrane phospholipids by phospholipase A2. Arachidonic acid is then converted into prostaglandin H2 (PGH2) by cyclooxygenases (COX). The metabolism of PGH2 to PGE2 is catalyzed by prostaglandin E synthase (PGES). While COX-1, cytosolic PGES and microsomal PGES-2 (mPGES-2) are constitutively expressed, COX-2 and mPGES-1 are induced in response to various pro-inflammatory and mitogenic stimuli1PGE2 functions by acting on one of the four E prostanoid (EP) receptors, EP1-4, all belonging to the rhodopsin-type G-protein-coupled membrane receptor family2+ T cell responses. By down-regulating IL-12 expression in antigen presenting cells (APC), it creates a cytokine milieu which favors the development of Th2 cells while suppressing Th1 differentiation1315et al. have demonstrated that EP2/EP4-mediated activation of the cAMP-PKA pathway actually facilitates IL-12-driven Th1 differentiation. PGE2 is also involved in the regulation of Th17 differentiation. On one hand, PGE2 potently suppresses the development of Th17 cells from naïve T cells induced by IL-6 and TGF-β1519192021PGE2 displays pleiotropic actions in a wide array of tissues, ranging from the cardiovascular, neural, renal, respiratory, hematopoietic, to the immune system67891011The commitment of a B cell to an IgE-producing cell is critically dependent on a unique type of intrachromosomal deletional recombination called class switch recombination (CSR)2425in vivo. Over years, a number of studies have investigated the PGE2-mediated effect on various aspects of asthma pathology. Diverse and sometimes opposite effects are revealedIgE is thought to have evolved to provide protective immunity against helminth parasites and certain noxious substances2729in vivo, and the relevance of PGE2-regulated IgE production in the development of asthma. In the study of the mechanism underlying PGE2 enhancement of IgE production, we focused on potential modulatory effect of PGE2 signal on STAT6 and NF-κB activation induced by IL-4 and LPS, respectively.Using mice with targeted deletion of the gene encoding the EP2 receptor, we analyzed the contribution of EP2-mediated signal in PGE2-enhanced IgE production, the impact of EP2 deficiency on antigen-specific IgE responses in vivo.PGE2 displays a potent enhancing effect on IgE production by B cells in cultures192021IgE is believed to be implicated in the pathogenesis of asthma29+ cells generated from EP2-deficient B cells remained at similar levels in the presence or absence of PGE2 23We further addressed the possibility that EP2-mediated signal may affect other aspects of B cell activation, such as cell proliferation and survival, which in turn contributes to the reduced IgE production. As indicated by CFSE dilution, PGE2 caused a slight but significant inhibition of LPS-induced proliferation of wild type B cells. Such an effect was dependent on EP2 as it was not observed with EP2-deficient B cells . As muchTaken together, these results suggest that the EP2-mediated signal promotes IgE production most likely through enhancement of IgE class switch and development of IgE-secreting B cells.The mechanism by which PGE2 enhances IgE class switch remains elusive. Among the multiple factors involved in the regulation of IgE class switch, IL-4-STAT6 signaling is of particular importance as the levels of IgE are dramatically reduced in IL-4- and STAT6-deficient mice32In B cells, STAT6 regulates the expression of a wide array of genes, some of which are critically involved in IgE productionCD23, a negative regulator of IgE response, is another target gene of STAT6. Following IL-4 stimulation, STAT6 activity promotes the expression of CD23, which presumably constitutes a negative feedback mechanism for IL-4-induced IgE production24In addition to STAT6, the classical pathway to IgE switching involves NF-κB activity, for which both CD40 and LPS are potent inducersin vivo and contributes to the development of OVA-induced asthma.The present study explored the role of EP2-mediated signal in the regulation of IgE production and its pathological relevance in asthma. The major findings are briefly summarized as follows: 1) the EP2 receptor is required for PGE2-enhanced IgE production by B cells; 2) EP2-mediated signal increases IgE production by facilitating IL-4-induced STAT6 activation and transcription of downstream targets critical for IgE class switching; 3) PGE2 signaling through EP2 promotes antigen-specific IgE responses Previous analyses with EP-selective agonists suggest that both EP2 and EP4 receptors contribute to PGE-induced effect on B cellset al., in which STAT6 is suggested having a self-promoting potential on its own transcription2420IgE class switching is critically dependent on the transcription factors NF-κB and STAT6. Upon activation by CD40 ligation or LPS stimulation, NF-κB synergizes with STAT6, which is principally activated by IL-4 or IL-13, to induce Cε germline transcription and AID expression, two events essential for CSR to IgE37in vitro192021in vivo. Several studies monitored IgE levels in mice deficient for PGE2 synthesis4041et al. reported that deficiencies in neither Cox-1, Cox-2 nor mPGES1 affected the serum levels of IgE after immunization with OVAAlthough the enhancing effect of PGE2 on IgE production was well illustrated with purified B et al. examined disease development in mPGES-1-deficient mice using a Der f-induced allergic model+ T cells at the sensitization stageThe role of PGE2 in the pathogenesis of asthma has been intensively studied. The majority of early studies indicate a beneficial effect444640et al.Opposite to the heightened allergic responses reported by Zaslona In summary, the present study shows that PGE2-enhanced IgE production by B cells is mainly mediated by EP2, possibly through synergistic activation of STAT6. Loss of EP2 leads to a reduced IgE response to allergens and attenuated airway inflammation in the OVA-induced asthma model. These data not only reveal an important regulatory role of the PGE2-EP2-STAT6 signaling pathway in IgE response but also underscore the challenge in the development of therapeutic strategy targeting PGE2 and its receptor in allergic diseases.The methods were carried out in accordance with the approved guidelines of Peking University Health Science Center.The EP2 knockout mice was generated as previously describedMice were sensitized by intraperitoneal injections of 20μg of OVA  emulsified in 2 mg aluminum hydroxide  in a total volume of 200μl on days 1, 8, 15, and challenged with an aerosol instillation of 1% OVA in PBS for 30 min on days 19, 21, 23. Control animals received PBS only. Two days after the final challenge, lung mechanics were measured, and the lung, serum and bronchoalveolar lavage fluid (BALF) were collected for further analysis.Hyperresponsiveness of airways was measured by placing mice in a whole-body plethysmograph Immediately after intraperitoneal injection with an overdose of pentobarbital (50 mg/kg), 0.6 ml of ice-cold PBS was instilled into the lung via the tracheal cannula and recovered by gentle manual aspiration. BALF collected from 3 repeats  was pooled and centrifuged. The total number of inflammatory cells were counted using cell counting chamber and the differential cell counts were obtained following staining with Diff-Quik Stain reagent .The lung tissue was fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E) to examine cell infiltration or with periodic acid-Schiff (PAS) to measure mucus production.+ B cells were sorted using FACSAria (BD Biosciences) with a purity > 98%.Single cell suspensions were prepared from the spleen and the erythrocytes were depleted with the ACK lysis buffer. For surface staining, cells were incubated for 25 min at 4 °C with fluorescent-labeled monoclonal Ab specific for mouse CD23, B220, CD4, CD44 and/or CD62L . To detect CD23 expression, 0.05% EDTA was included in the staining buffer to suppress IgE binding. For intracellular staining, cells were first fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and then incubated with PE-conjugated anti-IgE . Appropriate isotype-matched Abs were utilized for compensation adjustment. Flow cytometric analysis was performed on FACSCalibur using CellQuest software (BD Biosciences). B2206 cells/ml in Opti-MEM  supplemented with 10% fetal calf serum (FCS). To induce IgE production, cells were stimulated with LPS , or LPS+IL-4  for 3–7 days in the presence or absence of PGE2 . Different concentrations of PGE2, ranging from 10−10 M to 10−6 M, were tested in preliminary experiments. PGE2 at 10−8 M resulted in significant enhancement of IgE production with minimal effects on cell survival and was therefore used for subsequent experiments.Purified B cells were cultured at 1 × 106 B cells were incubated at room temperature for 5 min in 1 ml of PBS containing 5 μM carboxy fluorescein diacetate succinimide ester . Cells were washed twice to remove free dye before being put into culture under various conditions as described above. After culture for 3 days, cells were monitored for CFSE dilution using flow cytometry.For proliferation assay, 5 × 10Purified B cells were cultured under various conditions for 3 days. Cell apoptosis was determined by staining with FITC-coupled Annexin V  and 7-amino-actinomycin D , followed by analysis on a FACSCalibur.Total IgE in the serum and culture supernatant was measured using OptEIA™ Mouse IgE ELISA Set (BD Biosciences) according to the manufacturer’s instruction. The concentration of IgE was calculated using a standard curve obtained with mouse IgE of known concentrations. To measure serum levels of OVA-specific IgE, serially diluted sera was added into 96-well plates pre-coated with anti-IgE , followed by incubation with biotinylated OVA . The bound biotinylated OVA was detected with horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems) using tetramethylbenzidine as substrate (Sigma-Aldrich). The absorbance was read at 450/490 nm.Aid forward, 5′-TGCTACGTGGTGAAGAGGAG-3′, reverse, 5′-TCCCAGTCTGAGATGTAGCG-3′; GLTε forward, 5′-GCACAGGGGGCAGAAGAT-3′, reverse, 5′-CCAGGGTCATGGAAGCAGTG-3′; PSTε forward, 5′-TTGGACTACTGGGGTCAAGG-3′, reverse, 5′-CAGTGCCTTTACAGGGCTTC-3′; Stat6 forward, 5′-TGAGGTGGGGACCAGCCGG-3′, reverse, 5′-GTGACCAGGACACACAGCGG-3′; Gapdh forward, 5′-TTCACCACCATGGAGAAGGC-3′, reverse, 5′-GGCATGGACTGTGGTCATGA-3′.Total RNA from cultured B cells was isolated with Trizol Reagent (Invitrogen) and was used to make cDNA oligo-dT primers and the Reverse Transcription System . For quantitative PCR, iQ SYBR Green Supermix  was used according to the manufacturer’s instructions. The amplification was performed on an iCycler (Bio-Rad Laboratories). The quantification was based on ΔCT and calibrated to the level in LPS+IL-4-treated wild type B cells. GAPDH was used as an internal control. Primers were used as follow: B cell cultured under various conditions were harvested and cell lysates were prepared in lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM aprotinin, 1 mM leupeptin, 1 mM EGTA, 1 mM Na3VO4, 1 mM tetrasodium pyrophosphate, and 10 mM NaF). The lysate was resolved on a 12% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with anti-STAT6 , anti-phospho-STAT6 , anti-NF-κB p65 , anti-phospho-p65 , or anti-β-actin , followed by probing with HRP-conjugated anti-rabbit antibody (Sigma Aldrich). The immunoreactive bands were detected by chemiluminescence with ECL detection reagents .P value less than 0.05 was considered as statistically significant.All experiments were repeated at least three times, with 4–6 mice for each group each time. Data are presented as mean ± the standard error of the mean (SEM) unless otherwise specified. For statistical analysis, unpaired Student’s t test was performed using GraphPad Prism software . A How to cite this article: Gao, Y. et al. Prostaglandins E2 signal mediated by receptor subtype EP2 promotes IgE production in vivo and contributes to asthma development. Sci. Rep.6, 20505; doi: 10.1038/srep20505 (2016).


Chondroitin sulfate proteoglycans (CSPGs) are widely expressed in the normal central nervous system, serving as guidance cues during development and modulating synaptic connections in the adult. With injury or disease, an increase in CSPG expression is commonly observed close to lesioned areas. However, these CSPG deposits form a substantial barrier to regeneration and are largely responsible for the inability to repair damage in the brain and spinal cord. This review discusses the role of CSPGs as inhibitors, the role of inflammation in stimulating CSPG expression near site of injury, and therapeutic strategies for overcoming the inhibitory effects of CSPGs and creating an environment conducive to nerve regeneration. The limited ability of the human central nervous system (CNS) to repair itself following injuries has been known since the days of Ancient Egypt 3,000–2,5000 BCE), as documented in the Edwin Smith papyrus . It had ,5000 BCEInjuries to the CNS can generally be classified into two overarching categories: traumatic and neurodegenerative. Traumatic lesions to the brain or spinal cord are largely contusive in nature and often result from falls, sharp blows, or sudden deceleration style injuries, rather than penetrating wounds , 8. UnliAmong the many CSPG molecules expressed in the CNS are the lectican group, which include aggrecan, three forms of versican , neurocan, and brevican . All memOther proteoglycans that have major roles in the pathology of CNS injury are phosphacan and NG2 . Phospha N-acetylgalactosamine and glucuronate disaccharide, the basic unit that composes the CS-GAG [ N-acetylgalactosamine  and glucuronate disaccharide (GlcA) of the GAG disaccharide, leading to the synthesis of the CS-A, CS-C, CS-D, and CS-E CS-GAGs (NAc and the 2 position of the GlcA [ In vitro experiments show that the disulphated GAGs (CS-D and CS-E) promote the growth of embryonic axons [The variations in the length of the core protein and the varying number of GAG side chains attached to the central domain are major determinants of the biological activity of an individual proteoglycan. However, the biological effects of a proteoglycan are also affected by the sulfation patterns of thee CS-GAG , 18. The CS-GAGs  [16–18, the GlcA . It has the GlcA –22. For the GlcA , 22 wherthe GlcA  and permthe GlcA . In vitric axons , 25. TakThe majority of CSPGs can interact with growth factors, cell adhesion molecules, and other ECM molecules in the local environment and may regulate their biological activity. CSPGs are widely distributed throughout the normal CNS, both during development and in the adult. CSPGs expression is especially rich in the embryonic brain and can direct cell migration and axonal outgrowth by providing guidance cues , 18. PhoVast strides have been made in characterizing and understanding the complex orchestration of biological events that occurs following a lesion to the CNS. Unfortunately, it is now widely known and accepted that the events occurring in brain or spinal cord tissue post-injury create an environment hostile to a regenerative response, resulting in the abortive nature of the reparative process , 28, 29.The extreme forces applied to the CNS during traumatic injuries result in disruption of axonal tracts, blood vessels, and glial cells located at the epicenter of the lesion . ImIn healthy CNS tissue, microglial cells exist in a nonactive or resting state, courtesy of microglia-neuronal communications –37. Howeβ) fibrils [β fibrils form senile plaques, which are the key diagnostic criteria of AD [While the etiologies of traumatic and degenerative lesions are different, during degeneration the CNS responds in a highly similar manner to that described for traumatic lesions, complete with reactive astrogliosis and the deposition of CSPGs. Hallmarks of Alzheimer's disease (AD) pathology include the formation of neurofibrillary tangles and extracellular accumulation of amyloid-beta .Parkinson's disease (PD) is a neurodegenerative movement disorder of unknown etiology, characterized by the selective loss of dopaminergic nigrostriatal neurons . PD patiα) is a key cytokine that may influence the progression of MS. TNF-α promotes the proliferation of bovine astrocytes and human astroglioma cell lines, which leads to reactive gliosis, as seen in active MS plaques [α also stimulates production of colony-stimulating factors from neighboring astrocytes, which act as chemoattractants for reactive cells. This results in increased migration of activated cells to sites of inflammation and increases in the proliferation and activation of microglia [Multiple sclerosis (MS) is a clinically heterogeneous demyelinating disease of the CNS , charact plaques . TNF-α aicroglia –72.β2-integrins, and leukocyte common antigen are upregulated, indicating the presence of reactive microglia in these tissues [+ leukocytes were CD8+ [Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, adult-onset disease that usually results in death within 5 years of its initiation. Clinical manifestations of the disease include initial muscle weakness and atrophy that progress to a spastic paralysis resulting from motor neuron degeneration . About 2 tissues , 77, 78.ere CD8+ , suggestere CD8+ .Trauma-induced CNS injury and autoimmune/neurodegenerative CNS disorders have several important similarities and differences in terms of the immune/inflammatory response. This observation was noted by Popovich and colleagues  comparinBoth traumatic and autoimmune/neurodegenerative CNS injuries show microglial activation and immune cell infiltration. Even the temporal sequence of events is similar in these scenarios, where microglia are recruited first, releasing inflammatory cytokines and reactive oxygen species (ROS), as well as upregulating their antigen presenting cell capabilities. Following increased BBB permeability, hematogenous macrophages, neutrophils, and lymphocytes follow, mediating myelin vesiculation, lipid peroxidation and further release of proinflammatory agents and free radicals. Demyelination ensues, resulting from oligodendrocyte injury and edema. However, in SCI this process is restricted to CNS myelin, whereas in conditions like MS, myelin destruction and ROS are found in the CNS as well as in the periphery .Increases in pro- and anti-inflammatory cytokine levels and ROS production are a common occurrence in traumatic and autoimmune or neurodegenerative CNS disorders. In terms of therapeutic approaches, this has been targeted repeatedly using a variety of strategies that include antibody treatments, administration of cytokine receptor antagonists, or even proinflammatory cytokines themselves –90. GlucOne of the main differences between SCI and autoimmune/neurodegenerative disorders is the induction time for cytokine expression. Trauma-induced CNS injury is characterized by a transient increase in proinflammatory cytokine levels that is followed by a relatively rapid restoration of baseline levels. In this instance, cytokines have a key role in the acute-phase response. Trauma related degeneration can be attributed to the initial trauma itself, since cell death and tissue necrosis occur as early as one hour after injury. On the other hand, neurodegenerative conditions like MS and ALS owe their prolonged progression to a slow and persistent increase in proinflammatory cytokines. Degenerative processes seen in these cases are more likely to be the result of direct effects of cytokines .γ), and TNF-α are all known and documented to be proinflammatory and found to be upregulated in cases of CNS tissue damage [α, IFN-γ, were injected into normal brain tissue, a significant amount of reactive astrogliosis was observed around the injection sites [α; a cytokine expressed by macrophages) and transforming growth factor beta  resulted in the upregulation and synthesis of chondroitin 6-sulphate proteoglycans in brain tissue and in vitro cell culture experiments [As previously noted, following any type of insult to the central nervous system, an immune response is elicited and a combination of vascular macrophages and active microglia infiltrate the lesion site. It is well documented that vascular macrophages and activated microglia synthesize and secrete many different proinflammatory cytokine and chemokine molecules –99. Actie damage , 100. Inon sites , 94, 96.eriments , 97. Interiments .CSPGs are upregulated rapidly in the tissue surrounding a lesion site, due to the induction of reactive gliosis see . There aμm to 500 μm around a spinal cord lesion. Brevican lies close to the immediate injury site as well, deposited within 300 μm of the lesion border. Phosphacan and versican are expressed in a more broad range deposition, extending in a diffuse pattern as far as 600 μm from the impact site [In traumatic lesions, CSPGs can be observed at the lesion very quickly, within the first 24 hours. However, the temporal pattern in which individual CSPG molecules are produced is different. Neurocan is the first to appear, with brevican and versican following. Their expression levels are maximal two weeks after injury, in a spinal cord injury model . Peak exact site . Additioact site  followinact site . While tβ-amyloid plaques and neurofibrillary tangles was examined, it was found that these lesions were surrounded in a shell of GFAP positive reactive astrocytes, which indicate that reactive gliosis had occurred [In neurodegenerative lesions, the timeline of CSPG expression is not well understood, as all histopathology in humans occurs post-mortem. However, several studies have documented the presence of CSPGs in human tissue and upregulation in animal models of disease. In Alzheimer's disease, when the formation of the occurred . Furtheroccurred , 107. Thoccurred . Furtheroccurred . In otheoccurred , 111.Regardless of the etiology, most CNS lesions involve an immune response, including the recruitment of vascular macrophages and activated microglial cells , 113. Th in vitro studies have demonstrated that CSPGs can interact with adhesion molecules expressed on various cell types [ in vitro that CSPGs can influence the activity of the axonal growth cone. In dorsal root ganglion cultures, the presence of CSPGs induced changes in local protein synthesis in the growth cone, with an increase in RhoA transcripts  being found locally within the growth cone after CSPG contact [At the molecular level,ll types . When axll types , 33. Thell types . It has  contact . In vitro, purified brevican has been shown to be inhibitory to both axonal attachment and growth [ in vitro and are not inhibited by the presence of purified versican [ in vitro studies have demonstrated that NG2 can be inhibitory to the process of axonal outgrowth, while other studies have demonstrated that NG2 is not only permissive to axonal growth but can stabilize the axon post-injury [While CSPGs are widely accepted to be inhibitory to axonal regeneration, the inhibitory nature of individual CSPG molecules varies among the proteoglycans.d growth , 116, whd growth , 116. Coversican , 118. Cet-injury , 119–122t-injury , and it t-injury , 124. Th in vitro model of the glial scar with isolated OPCs revealed a significant inhibition of process outgrowth and differentiation [While the inhibitory effects of CSPGs on axons have been known for some time, the effects they exert on other cell populations, such as oligodendrocytes, have only been recently considered. CSPGs exert a highly inhibitory influence on oligodendrocytes –127. Stuntiation . When thntiation , 127.In vivo, CSPG expression can modulate the migration and differentiation of endogenous OPCs that are attracted to CNS lesions. Accumulation of OPCs at the edge of a lesion can be observed after spinal cord injury, in the regions distal to the lesion site [ion site –130. Theion site , 132. Inion site , 134. Thσ). Loss of the PTPσ receptor by gene knockdown or inhibition of receptor activation renders neuronal process outgrowth insensitive to CSPG deposits [σ receptor may also be involved with the observed inhibitory effects CSPGs exert on oligodendrocytes [σ has been observed in both neurons and oligodendrocytes, other receptors for CSPGs have been identified in neurons only, including Nogo 66 Receptor 1 (NgR1), Nogo 66 Receptor 3 (NgR3), and leukocyte common antigen receptor (LAR) [The cellular receptors which interact with proteoglycans have only recently been identified. Potential surface receptors for proteoglycans on neurons and glial cells include the protein tyrosine phosphatase sigma  , 137. To Proteus vulgaris, which catalyzes the removal of the glycosaminoglycan side chains from the central core protein [ in vivo studies have shown that by treating a CNS lesion site with the enzyme cABC, axonal sprouting, growth, and plasticity are significantly increased [Given that CSPGs are a major barrier to repair and regeneration, a key experimental strategy for enhancing axonal regeneration and plasticity has been to modify the inhibitory extracellular environment. The bacterial enzyme chondroitinase ABC (cABC) can neutralize the inhibitory nature of the CSPG molecules and thus was tested in many injury models. cABC is an enzyme produced by the bacteria protein . Numerouncreased –147. Thi in vivo, with digestion of CSPGs. To avoid the use of viral vectors, we have incorporated cABC into biodegradable nanospheres, which protects the enzymatic activity for many months [ in vivo model of spinal cord injury, the slow release of active cABC from nanospheres resulted in a significant increase in the level of CSPG digestion at 2 weeks and 1 month after injury when compared to direct injections, as well as a significant increase in the level of axonal sprouting throughout the lesion site [One critical issue with the use of cABC as a therapeutic agent is the thermal instability of the enzyme. The biological activity of cABC decreases quickly when in solution and is sensitive to temperature. A study by Tester et al.  demonstry months . When teion site .While cABC treatment digests CSPGs, there is evidence that following deglycanation, intact CSPGs are eventually reconstituted in the tissue. This turnover occurs approximately two-weeks following deglycanation and was demonstrated in a study by Crespo et al. . In thisWhile many studies claim functional recovery is a result of axonal regeneration, they fail to rule out other adaptive mechanisms that may account for the recovery of function. Such mechanisms include possible spontaneous remyelination of spared axons or the formation of alternative neural circuits –160. It Most studies using cABC have focused on the effects on neurons, documenting significant axonal sprouting and growth around a lesion after cABC treatment –144. HowIt is also possible that what is sometimes characterized as axonal regeneration may actually be the sprouting of spared axons . When th in vitro and in vivo, and this may affect remyelination near a CNS lesion. There is significant OPC infiltration to a SCI lesion site after cABC treatment, which occurs quickly after injury [ in vitro assay; the strongest inhibition was observed with the mixes of CSPGs containing high levels of both neurocan and phosphacan. This is highly homologous to the CSPGs composition of the glial scar [CSPGs have a strong inhibitory influence on OPC process outgrowth and differentiation, bothr injury , 162. Thial scar . Cellulaial scar , 128.Using a spinal cord injury model, treatment with cABC showed a significant increase in the number of OPCs found inside and around the lesion site . This ocIn the field of MS research, it has been demonstrated that inhibiting the synthesis of CSPGs improves the outcome of the pathology . Xylosid in vitro experiments have successfully targeted the enzymes responsible for the polymerization of the GAG side chains [Alternatively,e chains . When sie chains . In anote chains . Disrupt any insult to the CNS, traumatic or degenerative, an inflammatory reaction occurs, and the activation of microglia, astrocytes, and invasion of vascular macrophages result in an upregulation and synthesis of CSPGs (Significant progress has been made in understanding the post-injury tissue response after CNS injury, especially the identification and characterization of molecules at the lesion site that inhibit axonal regeneration, and identifying agents that can enhance the capacity for repair. There is overwhelming evidence that followingof CSPGs . There iModification of CS-GAG expression on CSPGs, while not completely ablating the CSPG molecule, is a reasonable approach to facilitating repair. One promising avenue is the administration of cABC at the site of a CNS lesion to remove the inhibitory GAG chains and neutralize the inhibitory nature of CSPGs. cABC has been tested in many injury studies and often results in an improvement in motor function. However, long distance axonal regeneration is rarely observed. In the SCI field, it is widely accepted that cABC will need to be used in conjunction with agents such as neurotrophins that can directly promote neuronal survival and stimulate axonal growth. An alternative strategy targets the new synthesis of CSPGs after injury by blocking the addition of GAG side chains. These agents also reduce the presence to the inhibitory GAG chains, but they have not been as well documented in injury models. Like cABC, it is probable that they need to be utilized in conjunction with factors that directly stimulate axonal sprouting and regrowth to maximize repair and recovery of function.While modification of CSPG expression is an attractive therapeutic approach, there are still many questions which need to be answered. How long would an agent such as cABC be needed? Axonal growth is a slow process, which can take months in animals and longer in humans; however, cABC may only be needed at specific times to allow the regeneration process to initiate. Delivery methods are very important as well. cABC is a labile enzyme, losing activity quickly in solution. There are several choices for long term delivery including viral expression and biomaterial packaging. However, how would this be silenced once regeneration is accomplished and cABC is no longer needed? Excess cABC would destabilize perineuronal nets in uninjured tissue, which could potentially result in aberrant axonal sprouting and circuit formation as well as synaptic instability. Although there are no reports of adverse effects in animal studies, there is also the possibility that the long term presence of cABC could trigger an immune response in humans.The most important consideration for repair of the damaged CNS is that controlling CSPG expression is only one aspect of the solution. Neurotrophic agents that directly promote neuronal survival and axonal growth are just as important, particularly for regeneration of specific neuronal populations, as are methods to direct these regenerating axons to their targets. CSPG regulation is but one important step in restoring CNS function after injury or disease.


Salvia hispanica L.) seed was hydrolyzed with an Alcalase-Flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut-off membranes . ACE-I inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions. The hydrolysate was extensive (DH = 51.64%) and had 58.46% ACE-inhibitory activity. Inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the <1 kDa fraction (69.31%). This fraction's amino acid composition was identified and then it was purified by gel filtration chromatography and ACE-I inhibition measured in the purified fractions. Amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ACE-I inhibitory strength, probably by blocking angiotensin II production. Inhibitory activity ranged from 48.41% to 62.58% in the purified fractions, but fraction F1 (1.5–2.5 kDa) exhibited the highest inhibition . The results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using Alcalase-Flavourzyme sequentional system.Synthetic angiotensin I-converting enzyme (ACE-I) inhibitors can have undesirable side effects, while natural inhibitors have no side effects and are potential nutraceuticals. A protein-rich fraction from chia ( Cardiovascular disease (CVD) affects the heart and blood vessels and is the principal cause of death worldwide. Considered the primary risk factor for CVD, high blood pressure, or hypertension, consists of a sustained increase in blood pressure levels. In 2000, >25% of the population worldwide (approximately 1 billion) suffered from hypertension, a figure predicted to increase to 1.56 billion by 2025 [Angiotensin I-converting enzyme  belongs to the class of zinc proteases that require zinc and chloride for activation. ACE plays an important role in blood pressure regulation via the renin-angiotensin system (RAS) and the kallikrein-kinnin system (KKS). In the KKS, ACE inactivates the vasodilator bradykinin, while in the RAS, ACE acts as an exopeptidase cleaving His-Leu from the C-terminal of decapeptide angiotensin I and producing the potent vasoconstrictor octapeptide angiotensin II .β-conglycinin and glycinin were hydrolysed by an acid proteinase from Monascus purpureus. The degree of hydrolysis and inhibitory activities of angiotensin I-converting enzyme (ACE) increased with increasing proteolysis time [Use of enzyme technologies for protein recovery and modification has led to production of a broad spectrum of food ingredients and industrial products. Hydrolysis selectivity is commonly manipulated by employing proteases from different sources due to their specificity for peptide bonds adjacent to certain amino acids. Enzymatic hydrolysis of food proteins is an efficient way to recover potent bioactive peptides . ACE-I isis time .Salvia L. belongs to the Lamiaceae family and includes about 900 species found worldwide, most mainly in the Mediterranean, Southeast Africa, and Central and South America. Cultivated for culinary, medicinal, and ornamental uses, Salvia species form part of ethnopharmacological traditions and are an important crop, especially for small farmers [Salvia hispanica protein rich fraction hydrolyzed with an Alcalase-Flavourzyme sequential system, and in ultrafiltered fractions from this hydrolysate.Population growth and shrinking food resources are ongoing challenges in developing countries, while excessive animal protein intake and associated unhealthy levels of saturated fats exposure are increasingly common in developed countries. In response, research interest has steadily grown in the search for new sources of proteins from nonconventional raw materials. The genus  farmers . Recent S. hispanica, L.) seeds were obtained in Yucatan state, Mexico. Reagents were of analytical grade and purchased from J. T. Baker , Sigma , Merck , and Bio-Rad . Angiotensin-converting enzyme from rabbit lung (2 units/mg protein) was purchased by Sigma . The Alcalase 2.4 L and Flavourzyme 500 MG enzymes were purchased from Novo Laboratories . Chia (μm screen) and a Ro-Tap agitation system. Flour was produced from 6 kg chia seed by first removing all impurities and damaged seeds, crushing the remaining sound seeds , and milling them . Oil extraction from the milled seeds was done with hexane in a Soxhlet system for 2 h. The remaining fraction was milled with 0.5 mm screen . The defatted chia flour was dried in a Labline stove at 60°C for 24 h. Extraction of the protein-rich fraction was done by dry fractionation of the defatted flour according to Vázquez-Ovando et al. . Briefly−1 for Alcalase and 50 LAPU g−1 for Flavourzyme; pH, 7 for Alcalase and 8 for Flavourzyme and temperature, 50°C. Hydrolysis was done in a reaction vessel equipped with a stirrer, thermometer, and pH electrode. In all treatments, the reaction was stopped by heating to 85°C for 15 min, followed by centrifuging at 9880 ×g for 20 min to remove the insoluble portion [The chia protein-rich fraction was hydrolyzed in batches by sequential treatment with Alcalase and Flavourzyme. A predigestion with Alcalase for 60 min was followed by incubation with Flavourzyme for 90 min. Hydrolysis conditions were substrate concentration, 2 g/100 g; enzyme/substrate ratio, 0.3 AU gh/htot∗100, where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolyzed bonds. The htot factor is dependent on the raw material amino acid composition and was determined by reverse-phase high performance liquid chromatography (RP-HPLC) [−1 HCl, placed in hydrolysis tubes, and gassed with nitrogen at 110°C for 24 h. They were then dried in a rotavapor and suspended in 1 mol L−1 sodium borate buffer at pH 9.0. Amino acid derivatization was performed at 50°C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed-phase column  and a binary gradient system with 25 mmol L−1 sodium acetate containing (A) 0.02 g L−1 sodium azide at pH 6.0 and (B) acetonitrile as solvent. Flow rate was 0.9 mL min−1, and elution gradient was time 0.0–3.0 min, linear gradient A : B (91 : 9) to A–B (86 : 14); time 3.0–13.0 min, elution with A–B (86–14); time 13.0–30.0 min, linear gradient A–B (86 : 14) to A–B (69 : 31); time 30.0–35.0 min, elution with A–B (69 : 31).Degree of hydrolysis (DH) was calculated by determining free amino groups with o-phthaldialdehyde following the methodology described by Nielsen et al.  as folloRP-HPLC) . SamplesThe hydrolysate was fractionated by ultrafiltration , using aμmol potassium phosphate buffer (pH 8.3), 300 μmol sodium chloride, 40 μmol 3% HHL in potassium phosphate buffer (pH 8.3), and 100 mU/mL ACE-I. This mixture was incubated at 37°C/45 min and the reaction terminated by addition of TT (3% v/v) in dioxane and 3 mL 0.2 M potassium phosphate buffer (pH 8.3). After centrifuging the reaction mixture at 10,000 ×g for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. All runs were done in triplicate. ACE-I inhibitory activity was quantified by a regression analysis of ACE-I inhibitory activity (%) versus peptide concentration, and IC50 values  was defined and calculated as follows:A represents absorbance in the presence of ACE-I sample, B absorbance of the control, and C absorbance of the reaction blank.ACE-I inhibitory activity in the hydrolysate and its UPF was analyzed following the method of Hayakari et al.  which isb is the intersection and m is the slope. Consider the following:−1 HCl, placed in hydrolysis tubes, and gassed with nitrogen at 110°C for 24 h. They were then dried in a rotavapor and suspended in 1 mol L−1 sodium borate buffer at pH 9.0. Amino acid derivatization was performed at 50°C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed-phase column  and a binary gradient system with 25 mmol L−1 sodium acetate containing (A) 0.02 g L−1 sodium azide at pH 6.0 and (B) acetonitrile as solvent. Flow rate was 0.9 mL min−1, and the elution gradient was time 0.0–3.0 min, linear gradient A : B (91 : 9) to A–B (86 : 14); time 3.0–13.0 min, elution with A–B (86–14); time 13.0–30.0 min, linear gradient A–B (86 : 14) to A–B (69 : 31); time 30.0–35.0 min, elution with A–B (69 : 31).Amino acid composition was determined in the UPF with the highest biological activity, according to the method of Alaiz et al.  Samples 12 (1.35 kDa), and Thr-Gln (0.25 kDa). Fractions selected for further peptide purification were pooled and lyophilized before RP-HPLC. After filtration through 10, 5, 3, and 1 kDa membranes in a high performance ultrafiltration cell, 10 mL of the fraction with highest ACE-I inhibitory activity was injected into a Sephadex G-50 gel filtration column (3 cm × 79 cm) at a flow rate of 25 mL/h of 50 mM ammonium bicarbonate (pH 9.1). The resulting fractions were collected to assay ACE-I inhibitory activity . Peptidein vitro ACE-I inhibitory activity and a Duncan multiple range done to identify differences between treatments. All analyses were done according to Montgomery [All results were analyzed in triplicate using descriptive statistics to estimate means and variation. One-way ANOVAs were run to evaluate ntgomery  and procBacillus licheniformis and the Flavourzyme (EC 3.4.11.1) is a fungal protease from Aspergillus oryzae with both endo- and exopeptidase activities. The bacterial endoprotease Alcalase is limited by its specificity, resulting in DHs no higher than 20%–25%, depending on substrate, but can attain these DHs in a relatively short time under moderate conditions. When Alcalase is used to hydrolyze protein it tends to produce peptides whose C-terminals are amino acids with large side chains and no charge , such as Ile, Leu, Val, Met, Phe, Tyr, and Trp. The hydrolysis process is accelerated when peptide N-terminals have hydrophobic amino acids [With a protein content of 46.7%, the chia protein-rich fraction proved to be good starter material for hydrolysis. Production of extensive  hydrolysates requires use of more than one protease because a single enzyme cannot achieve such high DHs within a reasonable time period. For this reason, an Alcalase-Flavourzyme sequential system was used in the present study to produce an extensive hydrolysate. Alcalase (EC 3.4.21.62) is a proteinase from no acids . The funno acids .S. hispanica is an appropriate substrate for producing extensive hydrolysates (DH higher than 10%), and a natural source of peptides with potential bioactivity.Chia hydrolysates obtained with the Alcalase-Flavourzyme sequential system had clear biological activity and are promising prospects for use in new product development. The highest DH in the present study (51.6%) was attained with Flavourzyme at 150 min. However, it was made possible by Alcalase predigestion, which increases the number of N-terminal sites, thus facilitating hydrolysis by Flavourzyme. This DH was higher than reported for hard-to-cook bean hydrolyzed with Alcalase-Flavourzyme (43%) or pepsin-pancreatin (26.2%) for 90 min . But it A number of natural ACE-I inhibitors have been isolated from different organism proteins, including peptides extracted by enzymatic hydrolysis. When added to food systems, enzymatic hydrolysates have exhibited advantages such as improved water-binding capacity, emulsifying stability, protein solubility, and nutritional quality. Enzymatic hydrolysis has become a valuable tool for modifying protein functionality . During Amaranthus hypochondriacus) albumin 1 and globulin [The protein hydrolysate obtained from the chia protein-rich fraction had 58.5% ACE-I inhibition. This is lower than the 79.5% reported for yak milk casein hydrolyzed with Alcalase for 240 min , but higglobulin .P < 0.05) in activity in progressively smaller fractions; that is, the >10 kDa fraction had the lowest activity and the <1 kDa had the highest. Use of ultrafiltration membranes clearly helped to enrich specific peptide fractions. This coincides with a study in which the <1 kDa fraction of an Alaska pollock  frame protein hydrolysate had the highest ACE-inhibitory activity  [50 = 280 μg/mL) than its corresponding <5000 kDa ultrafiltered fraction (IC50 = 190 ± 5 μg/mL) [Ultrafiltration of the hydrolysate produced UPFs with increased biological activity. ACE-I inhibition ranged from 53.8% to 69.3% , with cl7 μg/mL) . In anot5 μg/mL) . A great5 μg/mL) . When taAn amino acid profile was generated for the <1 kDa UPF because it had the highest ACE-I inhibitory activity. During hydrolysis, asparagine and glutamine partially converted to aspartic acid and glutamic acid, respectively; the data for asparagine and/or aspartic acid were therefore reported as Asx while those for glutamine and/or glutamic acid were reported as Glx. The high inhibitory activity (69.3%) exhibited by the <1 kDa UPF was probably due to its high concentration of hydrophobic amino acids (41.68 g/100 g), including Pro (6.11 g/100 g), Phe (11.03 g/100 g), Leu (10.23 g/100 g), and Ile (6.57 g/100 g) .Amino acid C-terminal hydrophobicity has the greatest influence on ACE-I inhibitory activity, and the higher the hydrophobicity the higher the inhibitory activity . This coAlthough the structure-activity relationship of ACE-I inhibitory peptides has not yet been established, these peptides show some common features. Studies of the structure-activity relationships in different ACE-I inhibitory peptides indicate that binding to ACE is strongly influenced by substrate C-terminal tripeptide sequence. Ondetti and Cushman  proposed50 value for F1 (3.97 μg/mL) was lower than those of gel filtration (Sephadex G-25) peptide fractions from tuna broth hydrolysate  [Fagopyrum esculentum Moench . Other studies suggest that ACE inhibition by hydrolysates depends on the source species and hydrolysate purity level. For instance, production of a 1–3 kDa protein hydrolysate from Alaska pollock frame using an ultrafiltration membrane bioreactor system resulted in high ACE-I inhibition (IC50 = 110 μg/mL), but further purification using consecutive chromatographic methods in a SP-Sephadex C-25 column and HPLC in an octadecylsilane column resulted in a still stronger effect (IC50 = 14.7 μg/mL) [Sepia officinalis) muscle protein was found to have high ACE-I inhibitory activity (87.1 ± 0.9% at 200 μg/mL). However, size exclusion chromatography with a Sephadex G-25 produced a fraction (P6) with yet higher ACE-I inhibition (IC50 = 11.6 μmol/L), which, when fractionated by reverse-phase (RP)-HPLC, was found to contain Ala-His-Ser-Tyr, Gly-Asp-Ala-Pro, Ala-Gly-Ser-Pro, and Asp-Phe-Gly [Of the UPFs, the <1 kDa fraction exhibited the highest ACE-I inhibitory activity and was selected for further fractionation. A molecular weight profile was generated of this UPF using gel filtration chromatography (Sephadex G-50 column). This profile was typical of a protein hydrolysate formed by a pool of peptides, with gradually decreasing molecular masses. Elution volumes between 406 and 518 mL included free amino acids and peptides with molecular masses ranging from 0.4 to 3.6 kDa. This range was fractionated into three fractions and ACE-I inhibitory activity determined for each. Fractions with elution volumes smaller than 406 mL and greater than 518 mL were not analyzed because they largely included peptides with high molecular weights, as well as free amino acids. ACE-I inhibitory activity (%) in the <1 kDa UPF ranged from 48.4% to 62.6% . The hig0 μg/mL)  or from 7 μg/mL) . In anot-Phe-Gly .in vivo studies are needed to confirm the effect of these peptides since it is both difficult and unwise to extrapolate directly from in vitro to in vivo activity. The main challenges in doing this are determining bioavailability of ACE-I inhibitory peptides after oral administration and the fact that peptides may influence blood pressure by mechanisms other than ACE-I inhibition. To exert an antihypertensive effect after oral ingestion, ACE-I inhibitory peptides must reach the cardiovascular system in an active form, meaning that they need to remain active during digestion by human proteases and transport through the intestinal wall into the blood. Bioavailability has been studied for some ACE-I inhibitory peptides, and it is known that proline-containing peptides are generally resistant to degradation by digestive enzymes. Peptides can be absorbed intact through the intestine by paracellular and transcellular routes, although postabsorption bioactivity potency is inversely correlated to chain length [In ACE-I inhibitory peptides from chia, the protein-rich fractions are not as potent as hypertension treatment drugs but hold promise as a safe, natural therapeutic agent without adverse side effects. The potential of chia protein-derived peptides as antihypertension agents depends on the ability of these peptides to reach their target site without suffering degradation and consequent inactivation by intestinal or plasma peptidases. Resistance to peptidase degradation is a probable prerequisite for any ACE inhibitory hydrolysates/peptides to exercise an antihypertensive effect after oral or intravenous administration. Proline-containing peptides are generally resistant to degradation by digestive enzymes , and trin length .50 values far higher than the 0.0013 μg/mL of Captopril, a synthetic ACE-I inhibitor [F < 1 kDa fraction had high ACE-I inhibitory activity, suggesting that ACE-I inhibitory peptides are rich in hydrophobic amino acids (aromatic or branched chains) and in proline. ACE-I inhibiting peptides from food sources have garnered increasing attention in recent years as promising natural biofunctional alternatives to synthetic drugs. Many of these peptides have been discovered in enzymatic hydrolysates of different food-source proteins and subsequently applied in the prevention of hypertension and initial treatment of mildly hypertensive individuals. The ongoing search for natural ACE inhibitors may eventually help to create safer and less costly alternatives to synthetic pharmaceutical treatments [All the chia derivatives studied here had ICnhibitor . Nonetheeatments .Chia proteins hydrolyzed with the Alcalase-Flavourzyme sequential enzyme system resulted in hydrolysates with ACE-I inhibitory activity. Ultrafiltration produced a very low molecular weight fraction (<1 kDa) which had the highest activity. The hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods. Enzymatic hydrolysis and ultrafiltration are promising bioprocesses for production of new bioactive food ingredients such as ACE inhibitory peptides purified from chia hydrolysate.


Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBP's bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional  The main target of the pharmaceutical industry for antihypertensive drugs remains angiotensin-converting enzyme (ACE). This transmembrane metalloprotease catalyzes the conversion of angiotensin (Ang) I to a potent vasoconstrictor Ang II in vivo BPL action via ACE inhibition, the inhibitory peptides must reach and remain in the target tissue in their active form. It has been reported that short-chain peptides are absorbed in the blood in relatively intact form in vivo.Many peptidic ACE inhibitors with blood pressure-lowering (BPL) effects have been identified in antihypertensive foods in vivo were responsible for BPL effects of these peptides, which were similar to the effects of neo-FBS; their specific BPL mechanisms remained unclear.In the previous study, we developed a new antihypertensive food called neo-fermented buckwheat sprouts (neo-FBS) by lactic bacteria fermentation of buckwheat sprouts in vitro and in vivo assays generally used to evaluate food-derived peptidic ACE inhibitors. In addition, we performed a new ex vivo ACE-inhibitory assay to investigate detailed BPL mechanism based on ACE inhibition by FBPs in living body tissue. The ex vivo assay measured FBP effects on ACE expressed on SMCs by using endothelium-denuded thoracic aorta ring of SHRs. Finally, we discussed the ACE-inhibitory properties of FBPs based on the in vitro, ex vivo, and in vivo results.In this study, we investigated the BPL mechanisms of these six BPL peptides isolated from neo-FBS (FBPs) by measuring vasorelaxation and ACE-inhibitory activity. Vasorelaxation was measured using the SHR thoracic aorta ring assay. The ACE-inhibitory activity was measured by the All animal experiments were performed with 10–13 week-old male SHRs (SHR/NCrlcrlj). Housing and feeding was conducted as previously described l-histidyl-l-leucine (Hip-His-Leu) was obtained from the Peptide Institute . Ammonium formate, borate, diethyl ether, ethyl acetate, formate, hydrochloric acid (HCl), HPLC-grade acetonitrile, sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), magnesium acetate (Mg(CH3COO)2), magnesium sulfate (MgSO4), Nonidet P-40, sodium hydrogen carbonate (NaHCO3), sucrose, and tris(hydroxymethyl)aminomethane were purchased from Kanto Chemical Co., Inc. . Trifluoroacetic acid (TFA) was purchased from Watanabe Chemical Industries, Ltd. .Acetylcholine chloride (ACh), Ang I (>97%), Ang II (>97%), captopril, and phenylephrine hydrochloride (PE) were purchased from Wako Pure Chemical Industries . ACE (EC 3.4.15.1) purified from the rabbit lungs was obtained from Sigma-Aldrich Japan K.K. . Hippuryl-Six FBPs  were recently identified in neo-FBS by HPLC analysis, LC-MS/MS, and Edman degradation 2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, pH 7.4) bubbled with 95% O2/5% CO2 and stretched to a resting tension of 1.5 g. Equilibration of the tension, confirmation of the endothelium function by Ach, and stabilization of the vasoconstrictive force by PE were performed as previously described The effects of FBPs were measured using thoracic aorta isolated from SHRs (n = 6). Isolation of the thoracic aorta and preparation of the aortic rings (2–3 mm long) were performed as described previously in vitro ACE inhibition assay was performed using the method described previously In vitro ACE-inhibitory activity (%) was calculated using the equation described previously 50). All measurements were performed in triplicate for each sample concentration, and the results were expressed as the mean ± S.E.The in vitro and BPL activity in vivo, of a food derived di-peptide Val-Tyr (VY) that affects renin-angiotensin system. This system plays an important role in the regulation of blood pressure and body fluid homeostasis, and VY is known as an ACE inhibitor with BPL activity 2+-ATPase inhibited by Ang II in vitro ACE-inhibitory activity (IC50) and minimum effective BPL dose of VY were 26.0 µM and 1.0 mg/kg, respectively in vitro ACE inhibitor was DVWY (690 µM); based on this concentration, the minimal in vivo dosage of FBPs for effective BPL via ACE inhibition was estimated as 10 mg/kg, which is consistent with the concentration of neo-FBS that induced a significant in vivo ACE inhibition in our previous study Animals were assigned to six FBP-treated groups and six control group; the average blood pressure was the same in each group. After being deprived of food for 12 h, rats were given an oral dose of 10 mg/kg BW FBP in purified water (n = 6) by gastric intubation. The dose, which was higher than the minimum effective BPL dose of FBP (0.1 mg/kg BW), had been estimated based on ACE-inhibitory activity 3COO)2, 25 mM sucrose, 50 mM tris(hydroxymethyl)aminomethane, 0.50% Nonidet P-40, pH 7.8) for 5 min  or 10 min (thoracic aorta). The homogenates were centrifuged at 12,000×g for 20 min at 4°C, and the supernatant was immediately used to measure tissue ACE activity. Plasma samples were prepared by centrifugation  of the blood collected in lithium heparin-treated tubes. Tissue and plasma ACE activity was measured as described for the in vitro assay and expressed as units/mg-protein or units/mL, respectively. Tissue protein content was measured by the BCA Protein Assay Reagent Kit . The results were expressed as the mean ± S.E.Prior to and 6 h after the administration, SBP and DBP were measured in all SHRs by the tail-cuff method ex vivo assay measured both Ang II production and Ang II-mediated vasoconstriction in endothelium-denuded thoracic aorta rings isolated from SHRs; the results were used to compare the effects of different FBPs and captopril on tissue ACE activity. Isolation of the thoracic aorta from SHRs (n = 12) and preparation of endothelium-denuded aortic rings was performed as described above. Endothelium was removed to eliminate the influence of endothelium-derived vasodilators. Prostanoids and nitric oxide, which are major endothelium-derived vasodilators, were shown to strongly suppress vasoconstriction Our Before the experiment, the influence of endothelial removal on Ang II production was evaluated using endothelium-intact and endothelium-denuded aortic rings by a procedure similar to that for the negative control described below.in vivo assay were tested because this assay evaluates tissue ACE-inhibitory activity in thoracic aorta rings. Captopril and Krebs solution were used as the positive and negative controls, respectively. After equilibrating and stabilization of vasoconstriction in aorta rings by PE, 2.0 mL of Krebs solution was removed from the organ bath to add test sample and Ang I. Then, 1.0 mL of DVWY, FDART, FQ, WTFR, captopril, or Krebs solution was added to endothelium-denuded ring tissues to reach final concentrations of 0.1, 1.0, or 10.0 µM for FBPs and 0.1, 0.5, or 1.0 µM for captopril; 1.0 mL of Krebs solution was added to endothelium-intact ring tissues; all the concentrations were determined in preliminary experiments. No significant vasoconstriction or vasodilation of the aorta rings was caused by FBPs. After 30 min incubation, 1.0 mL of 0.20 µM Ang I was added and the aortic tension was recorded. When vasoconstriction plateaued, Krebs solution in the organ bath was transferred to a test tube and heated at 90°C for 5 min to stop the enzyme reaction; the heat-treated samples were dried under vacuum and frozen at −80°C until UPLC-MS analysis. The aorta rings were used in the BCA assay to express Ang II production as pmol/mg-protein. To evaluate FBP suppressive effect on Ang II-mediated vasoconstriction, the tension was recorded and expressed as the percentage of maximum constriction of the negative control; the 50% effective concentration (EC50) was determined by plotting FBP and captopril concentrations against the percent of suppression.Four FBPs  with significant thoracic aorta ACE-inhibitory activity in the 2 gas flow (desolvation), 50 L/h N2 gas flow (cone), 100°C source temperature, and 350°C desolvation temperature. The mass spectrometer was operated in positive mode and selected ion monitoring (SIM) mode at m/z 523.8 [M+2H]2+ for Ang II. Ang II concentration was calculated using standard calibration curves in the linear range of 0.0010–1.0 µM with a correlation coefficient of 0.9993; linearity was described by the equation y = 69455x+423.12. The IC50 of Ang II production was determined by plotting FBP and captopril concentration versus inhibition of Ang II production calculated as the percentage of negative control group. For each sample, measurements were performed in triplicate; the results were expressed as the mean ± S.E.UPLC-MS analysis was performed to quantify Ang II produced from Ang I in thoracic aorta treated with FBPs or captopril. Vacuum-dried samples were dissolved in 500 µL acetonitrile/4.0 mM ammonium formate (5∶95 v/v) with 4.0 mM formic acid prior to UPLC-MS analysis, performed at the CREFAS ; Quattro micro API (MS) with an ACQUITY UPLC  was used. Separation was conducted at 35°C using a CHEMCOBOND 5-ODS-W reversed-phase column . Elution was performed at 0.80 mL/min using acetonitrile/4.0 mM ammonium formate (5∶95 v/v) with 4.0 mM formic acid (solvent A) and acetonitrile/4.0 mM ammonium formate (90∶10 v/v) with 4.0 mM formic acid (solvent B): 0–2 min, isocratic 0% solvent B; 2–10 min, 0–40% solvent B; 10–15 min, isocratic 40% solvent B; 15–20 min, 40–90% solvent B. UV detection was performed at 215 nm with an injection volume of 20 µL. Mass spectra were acquired in electrospray ionization (ESI) mode using 3500 V capillary voltage, 30 V cone voltage, 350 L/h Nin vitro ACE-inhibitory activities of FBPs were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. We assessed correlation between the suppression of Ang II production and Ang II-mediated vasoconstriction to validate the ex vivo ACE inhibition assay. For this, Pearson's correlation analysis widely applied in studies of enzyme inhibitory activity, was used. A coefficient of correlation (r) value >0.70 and P value (two-sided test) <0.05 were considered statistically significant. In all animal tests, Student's t-test was used to identify significant differences between each FBP group and control group .All results are expressed as mean ± S.E. The in vivo is underlined by vasorelaxation and ACE inhibition In our previous study, we found that the BPL effect of neo-FBS Vasorelaxant effects of FBPs were measured using PE-preconstricted endothelium-intact or endothelium-denuded thoracic aorta rings of SHRs (n = 3). DVWY, FDART, and VVG significantly dilated the endothelium-intact aorta rings , while Fin vitro assay with Hip-His-Leu substrate. in vitro (n = 3). Based on the IC50 values, the most potent FBP was DVWY , followed by FDART (1.9±0.1 mM), WTFR (6.7±0.5 mM), FQ (7.4±0.6 mM), VVG (39.6±5.7 mM), and VAE (55.9±1.9 mM). These experiments revealed significant variability among FBPs .FBPs were tested in a standard in vivo effects of FBPs on ACE inhibition at a dosage based on the in vitro activity (10 mg/kg BW). Although the dose was higher than the effective BPL dose of FBPs (0.1 mg/kg BW), it was lower than that estimated based on FBP weak in vitro ACE-inhibitory activity. SHRs were treated with each FBP and ACE activity was measured in the thoracic aorta, heart, liver, kidney, lung, and plasma 6 h after FBP administration . There was no significant difference in Ang II production between endothelium-denuded (55.5±3.5 pmol/mg-protein) and endothelium-intact samples (67.6±6.4 pmol/mg-protein), indicating that in thoracic aorta of SHRs, ACE is mainly expressed and acted on mesothelium. Thus, the endothelium-denuded aorta rings could be used to measure ACE-inhibitory activity of FBPs in thoracic aorta. 50 values were 0.15 µM (captopril), 5.64 µM (WTFR), 41.7 µM (FDART), 70.5 µM (DVWY), and 90.9 µM (FQ). These data suggest that FBPs effectively inhibited ACE in thoracic aorta at much lower concentrations than indicated by their in vitro activity (IC50).To examine specific ACE-inhibitory mechanisms of FBPs in living body tissues and their contribution to BPL activity, we designed a new ex vivo ACE inhibition assay, changes in vascular tone were also measured. 50 values were 0.75 µM (captopril), 36.8 µM (WTFR), 53.6 µM (FDART), 68.2 µM (DVWY), and 85.1 µM (FQ). These results confirmed that the decrease in Ang II production caused by FBPs suppressed the contractile response of SHR rings. Thus, we confirmed by the new ex vivo assay that in the thoracic aorta, FBPs inhibited ACE and suppressed vasoconstriction at low concentrations.In our new quantitative 50) as variable ‘x’ and suppression of vasoconstriction (EC50) as variable ‘y’ , indicating that the decrease in Ang II was directly related to the suppression of vasoconstriction without any interfering factors. Given that suppression of vasoconstriction results in BPL ex vivo results suggest that ACE inhibition by FBPs in the thoracic aorta effectively reduced blood pressure by suppressing Ang II-mediated vasoconstriction.The four FBPs reduced Ang II production by inhibiting ACE and suppressed Ang II-mediated vasoconstriction, thus triggering reduction in blood pressure. However, we could not conclude that the suppression of vasoconstriction was caused only by Ang II decrease. Although FBPs did not affect the endothelium-denuded PE-constricted thoracic aorta, they might demonstrate other vasoactive effects such as inhibition of Ang II binding to the AT1 receptor. To confirm that ACE inhibition was the major factor causing the suppression of vasoconstriction by FBPs, Pearson's correlation analysis was performed for ACE-inhibitory activity . Other BPL peptides IPP, VPP, and VY also inhibited ACE  https://hfnet.nih.go.jp/). Our results revealed that the in vitro activity of FBPs was much weaker than that of reported peptidic ACE inhibitors. Therefore, we measured the in vivo ACE-inhibitory activity of FBPs in SHRs treated with a dose of 10 mg/kg based on the concentration active in vitro.All FBPs showed ACE-inhibitory activity in vivo ACE-inhibitory activity of FBPs was the same level or greater than that of neo-FBS, indicating that FBP inhibitory activity was sufficient to cause BPL in SHRs. Indeed FBPs significantly reduced blood pressure in SHRs, especially SBP  was approximately 73,000 times weaker than that of captopril (IC50 0.092 µM) ex vivo WTFR activity (IC50 5.64 µM) was only 37.6 times weaker compared to captopril (IC50 0.15 µM) and the in vivo activity of WTFR was similar to that of captopril in vitro dwindled in the ex vivo assays and disappeared in vivo. The in vitro assay indicated that captopril bound ACE at a much lower concentration than FBPs. Although captopril IC50 values in vitro and ex vivo were comparable, FBP IC50 values ex vivo were much lower than in vitro. Tissue ACE is mainly expressed on the plasma membranes of SMCs in hypertensive state ex vivo assay, the incubation time of 10 min, which is one-third of proper incubation time (30 min), has waned the ACE-inhibitory activity of FBPs. The presence of FBPs, including WTFR, on thoracic aorta was qualitatively confirmed by UPLC-MS analysis using tissue homogenate prepared from the aorta rings after the end of the ex vivo assay (data not shown). By the ex vivo assay, we show that FBPs with higher tissue affinity might accumulate in the target organs until they can reach the threshold necessary for ACE inhibition at the level of captopril. In addition to tissue affinity, the in vivo ACE-inhibitory activity of FBPs is driven by high absorbability suggesting more efficient accumulation in target tissues compared to captopril. Considering all these factors, we hypothesize that high FBP bioavailability underlines their significant inhibitory effects on tissue ACE comparable to those of captopril.The Captopril is an effective medicine for cardiovascular diseases such as hypertension and heart failure. However, it strongly inhibits ACE in the lungs causing dry cough as a side effect. In addition, it is difficult to use captopril as a hypertension-preventive agent because its application is generally limited to the diseased patients. FBPs are constituents of neo-FBS which is lactic bacteria-fermented food prepared from buckwheat sprouts; the majority of tested FBPs (except for FQ, ex vivo assay was effective in evaluation of ACE-inhibitory mechanism with BPL action of peptidic ACE inhibitors in living body tissue.We found that the main BPL mechanism of FBPs was ACE inhibition in living body tissue. Additionally, we suggest that our novel