Moreover, co-immunoprecipitation and dual immunofluorescence studies confirmed ESR1, β-catenin, and lymphoid enhancer factor 1/T cell factor 3 co-localization in the nucleus in HESCs after E2 treatment. To determine the role of β-catenin signaling in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium from endometriosis patients into ovariectomized severe combined immunodeficiency mice. The implantation of the endometrium was suppressed by β-catenin siRNA. Collectively, studies regarding β-catenin signaling are critical for improving our understanding of the pathogenesis of estrogen-induced endometriosis, which can translate into the development of treatments and therapeutic strategies for endometriosis.Endometriosis is an estrogen-dependent disease that involves the adhesion, invasion, and angiogenesis of endometrial tissues outside of the uterine cavity. We hypothesized that a link exists between estrogen and beta-catenin (β-catenin) signaling in the pathogenesis of endometriosis. Human endometrial stromal cells (HESCs) were separated from eutopic endometrial tissues that were obtained from patients with endometriosis. β-catenin expression and cells invasiveness ability were up-regulated by 17β-estradiol (E A previThe aims of the present study were to investigate the molecular mechanism underlying the interaction between estrogen and β-catenin signaling in human endometrial stromal cells (HESCs), illustrate whether the β-catenin signaling pathway plays a critical role in estrogen-facilitated invasion and angiogenesis by HESCs, and determine the role of β-catenin in a non-obese diabetic severe combined immunodeficiency (NOD–SCID) mouse endometriosis model.All eutopic endometrial tissues were obtained from endometriosis patients by hysterectomy, which was performed at the Department of Obstetrics and Gynecology, Tongji Medical College, Union Hospital, Huazhong University of Science and Technology. The average age was 39 years (range 33–44) at the time of laparoscopy. The patients had not received any hormonal treatment for at least 6 months before the surgical procedure. Informed consent was obtained from each patient using protocols approved by the Human Investigation Committee at Tongji Medical College, Union Hospital, Huazhong University of Science and Technology. All samples were obtained in the proliferative phase of the cycle, which was confirmed histologically according to established criteria.g for 20 min to further remove leukocytes and erythrocytes, and the middle layer was collected and then washed with D-Hanks solution. The HESCs were placed in a culture flask and allowed to adhere for 24 h. The adherent stromal cells were cultured as a monolayer in flasks with DMEM/F12 containing 15% FBS, 20 mmol/l HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin and incubated in 5% CO2 at 37 °C. This method supplied 95% pure HESCs.Isolation and culture of HESCs began with the collection of eutopic tissues under sterile conditions. The tissues were then transported to the laboratory on ice in a 1:1 formula of DMEM/F12 (Gibco) with 10% fetal bovine serum . The minced eutopic endometrium was digested with collagenase type 2  for 30 min at 37 °C with constant agitation. The tissue pieces were filtered on a 100 μm wire sieve to remove debris. Following gentle centrifugation, the supernatant was discarded, and the cells were resuspended in DMEM/F12. The HESCs were separated from epithelial cells by passing them over a 400 μm wire sieve. The filtered suspension was layered over Ficoll and centrifuged at 1200 2 was purchased from Sigma–Aldrich (E-2758) and dissolved in DMSO. ICI 182 780 (ICI), an estrogen receptor antagonist was purchased from Cayman Chemical  and dissolved in DMSO. Cells were incubated with E2 at various doses for various times or with DMSO (control group).ECt values.Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Life Technologies), and 1 μg RNA was used for cDNA synthesis. SYBR Premix Ex Taq II  was used for PCR. Each reaction mixture consisted 5 μl 2× SYBR Premix Ex Taq II, 0.2 μM forward PCR primer, 0.2 μM reverse PCR primer, 0.2 μl 50× ROX reference dye, 1 μl cDNA, and 3.4 μl sterile distilled water in a final volume of 10 μl. Quantitative real-time PCR was performed using the preset PCR program of the StepOnePlus Real-Time PCR System  to quantify mRNA expression, with β-actin as an internal control. Primers for quantitative RT-PCR analyses were synthesized based on the GenBank database. The primers were as follows: β-catenin sense, 5′-AGCT TCCA GACA CGCT ATCA T-3′; β-catenin antisense, 5′-CGGT ACAA CGAG CTGT TTCT AC-3′; matrix metalloproteinase 9 (MMP9) sense, 5′-TGTA CCGC TATG GTTA CACT CG-3′; MMP9 antisense, 5′-GGCA GGGA CAGT TGCT TCT-3′; vascular endothelial growth factor (VEGF) sense, 5′-GAGG AGCA GTTA CGGT CTGT G-3′; VEGF antisense, 5′-TCCT TTCC TTAG CTGA CACT TGT-3′. The following PCR conditions were used: 95 °C for 10 s; 35 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s and a dissociation program of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. The expression levels of the target genes were calculated from the ΔΔg at 24 °C for 10 min after the cells were incubated for 30 min on ice. Cytoplasmic and nuclear proteins were extracted according to the instructions of a cytoplasmic and nuclear protein extraction kit . Cells were harvested in ice-cold PBS, scraped from culture dishes on ice using a plastic cell scraper, and collected in 1.5 ml micro-centrifuge tubes. The cytoplasmic lysis buffer contains 1 ml hypotonic buffer, 5 μl phosphatase inhibitor, 1 μl dl-dithiothreitol (DTT), and 10 μl phenylmethanesulfonyl fluoride (PMSF). The material was centrifuged at 3×1000 r.p.m. for 5 min to sediment the nuclei. The supernatant was then centrifuged at 12×1000 r.p.m. for 10 min to get the non-nuclear supernatant fraction. The nuclear pellet was then washed three times with cytoplasmic lysis buffer and resuspended in nuclear lysis buffer . The mixture was sonicated briefly to aid nuclear lysis. The nuclear lysates were collected after centrifugation at 12×1000 r.p.m. for 10 min at 4 °C. The samples were boiled for 10 min and then separated by 10% SDS–PAGE. The proteins were transferred to a PVDF membrane  in the presence of 20% methanol and 0.1% SDS. Nonspecific signals were blocked by incubating the membrane in 5% nonfat milk in Tris-buffered saline–Tween-20 for 1 h. The membranes were incubated with primary antibodies of interest overnight at 4 °C. The antibodies were directed against the following proteins: β-catenin , non-phospho β-catenin , GAPDH , or histone H1 . The blots were developed using HRP-conjugated secondary antibodies , and the proteins were visualized by an ECL procedure (Millipore) according to the manufacturer's recommendations.After the cells were washed with PBS, cell extracts were prepared in lysis buffer containing 40 mM Tris–HCl, 100 mM NaCl, and 0.1% Nonidet P-40 and supplemented with protease inhibitors. Whole-cell lysates were collected by centrifugation at 16000 2 at 37 °C.HESCs were grown in culture medium with 15% FBS before transfection. When the cells were 50–60% confluent, β-catenin siRNA  and Lipofectamine 2000 (Invitrogen) were added to opti-MEM, mixed, incubated for 20 min, and then added to the cells at room temperature (RT) according to the manufacturer's protocol. Scrambled siRNA (RiboBio) was used as a control. After 6 h, the mixture was replaced with phenol red-free DMEM/F12 with 15% FBS in 5% CORenilla luciferase expression plasmid, were purchased from GeneChem . Co-transfection experiments were performed in 24-well plates. In total, 1×105 cells were seeded per well in 500 μl medium. Then, 0.8 μg TOPflash or FOPflash and 0.008 μg pRL-SV40 reporter plasmid plus Lipofectamine 2000 transfection reagent were added to the HESCs. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Then, firefly luciferase activity was normalized to the corresponding Renilla luciferase activity.TOPflash, a TCF reporter plasmid; FOPflash, a negative control for TOPflash; and pRL-SV40, a 5 cells/ml was added to each upper compartment of the precoated units. After the cells were allowed to attach for 30 min, the units were transferred to wells containing 500 μl DMEM/F12 medium with 20% FBS as a chemoattractant and incubation was conducted for 24 h. After removing the cells and Matrigel from the upper surface of the membrane with a cotton bud and staining with 0.1% crystal violet for 15 min, the number of cells on the underside was determined using light microscopy . Five randomly selected fields were counted per insert.Transwell units with 8.0 μm pore-size polycarbonate filters  were precoated with 50 μl 1:2 diluted Matrigel (Sigma–Aldrich) and used to investigate cell invasion. A total volume of 100 μl suspension containing ∼10For co-immunoprecipitation (COIP) assays, cells were lysed in 400 μl lysis buffer (20 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% SDS, 0.5% deoxycholate, and protease inhibitors). Lysate aliquots (500 μg at 1 μg/μl) were precleared with 60 μl protein A-sepharose beads  for 1 h at 4 °C. Appropriate amounts of mouse-directed antibody against β-catenin , mouse antibody directed against estrogen receptor alpha , and mouse nonspecific IgG  were then added and incubated for 4 h at 4 °C. Preblocked agarose beads (100 μl) were added to the antibody/lysate mixture and incubated overnight at 4 °C. After the beads were washed three times with 1× PBS, the bound proteins were eluted in SDS sample buffer, resolved by SDS–PAGE, and analyzed by immunoblotting. The antibodies used for immunoblotting were rabbit directed antibodies against ESR1 , β-catenin , LEF1 , and TCF3 .2 dishes and treated with E2 (Sigma–Aldrich) at 10−8 mol/l for 48 h, with DMSO as a control. Then, the cells were fixed in ice-cold 4% paraformaldehyde for 10 min, washed with 1× PBS, and permeabilized in PBS containing 0.5% Triton X-100 for 3 min at RT. All cells were blocked  and incubated overnight (4 °C) with primary antibodies directed against β-catenin , LEF1 , TCF3 , and ESR1 . After the cells were exposed to the primary antibodies, they were incubated with Alexa Fluor 488- and 568-conjugated secondary antibodies and mounted with Prolong Gold antifade medium. Immunofluorescently labeled cell monolayers were visualized under an Olympus FV1000 laser scanning confocal microscope (Olympus).HESCs were cultured in 25 mm3 sections under sterile conditions. The fragments were cultured in phenol red-free DMEM/F12 (1:1)+10% FBS before transplantation into NOD–SCID mice (2 (0.2 μg/day) plus positive β-catenin siRNA (125 μg/kg per day) or scrambled siRNA (125 μg/kg per day) and saline solution control. All treatments were administrated via peritoneal injection. E2 was purchased from Sigma–Aldrich (Y0000852). Mice were sacrificed 10 and 21 days after implantation, the peritoneal cavity was examined, and tissue collected. The implanted endometrial lesions were harvested for hematoxylin and eosin staining and for immunohistochemical (IHC) analysis. Western blot analysis of the tissues was performed as described previously.Proliferative-phase eutopic endometrial tissues were obtained from ten premenopausal women with endometriosis during hysterectomy at Wuhan Union Hospital. Fresh endometrial tissue samples were fragmented into 2–3 mms.e.m. Unpaired t-tests and one-way ANOVA were used to demonstrate statistical significance. Statistical significance was set at P<0.05. All experiments were performed in triplicate.All values are shown as the mean±2 has been shown to stabilize β-catenin expression through estrogen receptor binding in neuronal cells , followed by maximal up-regulation at 10−8 mol/l (∼2.8-fold) and a decline at 10−6 mol/l (∼1.9-fold) while remaining high compared with the control (2 (10−8 mol/l) treatment caused β-catenin up-regulation (∼2.2-fold) at 24 h and that this up-regulation continued to increase until 48 h (∼2.9-fold). Next, we explored whether this E2-regulated response was reflected in the protein level of β-catenin. Western blot analysis demonstrated that β-catenin was up-regulated (∼4.1-fold) at 10−10 mol/l E2, then increased (∼4.5-fold) at 10−8 mol/l E2 but slightly declined at 10−6 mol/l E2 . Furthermore, we wanted to determine whether the estrogen-regulated responses are dependent on estrogen receptor (ESR). To address this question, HESCs were treated with ICI  in conjunction with E2 at various doses and times. We observed that the E2-regulated inductive responses of β-catenin mRNA and protein were indeed antagonized by ICI  or with control (DMSO) for 48 h. Western blot analysis indicated that E2 treatment led to an increase in β-catenin protein levels in the nucleus (∼2.6-fold) and to a slight decrease in β-catenin protein levels in the cytoplasm compared with the control (∼0.7-fold) (2 (∼3.67-fold) compared with the control. Next, we investigated the effect of E2 on TCF-induced transcriptional activity in HESCs. As shown in P<0.01) upon stimulation with E2 and could be abolished by ICI (**P<0.01). Collectively, these findings demonstrated that E2 activates β-catenin signaling in HESCs.Next, we wanted to confirm the effects of E.7-fold) E. As sho2 binds ER to stimulate target gene expression and a nongenomic mechanism in which E2 induces cellular effects independent of ER-related transcriptional activity  treatment, and the effect was maximal at 48 h. E2 stimulated ESR1 binding to β-catenin/TCF3/LEF1 in a time-dependent manner. Furthermore, β-catenin pulldown analysis detected an ESR1/TCF3/LEF1 complex. In contrast, the effects of ESR1, TCF3, and LEF1 interactions with β-catenin were maximal at 24 h (2 for 48 h. The expression of these proteins in the control (DMSO) group also showed a co-localization pattern, but the co-localization was less intense than that in the E2 group  and VEGF (∼4.2-fold) in HESCs, whereas ICI treatment significantly abrogated this phenomenon . All experimental groups were treated with E2 (10−8 mol/l) after transfection with siRNA. These data indicated that the E2-induced increase in the invasiveness of HESCs was dependent on β-catenin.After HESCs were treated with E*P<0.05; C. As sho2 increases the adhesiveness and invasiveness of endometrial lesions through β-catenin signaling in vivo, we established an animal model that would mimic ectopic implantation of the endometrium. To generate this model, human eutopic endometrial were implanted into the pelvic and peritoneal cavities of ovariectomized NOD–SCID female mice. Ten days after transplantation, endometriotic lesions were detected in 25% (two of eight) animals treated with E2 and scrambled siRNA and 75% (six of eight) of animals treated with E2 and β-catenin siRNA (2 and scrambled siRNA 21 days after transplantation (2 plus scrambled siRNA that developed viable endometriotic lesions was 63.6% (seven of 11 mice) after a 21-day incubation. No visible endometrial fragments were observed in mice treated with E2 plus positive β-catenin siRNA after a 21-day incubation or in the control groups after both 10- and 21-day incubations. The lesions were highly vascularized as observed on gross morphological examination  and MMP9 (∼2.4-fold) expression was observed in the endometriotic lesions from mice treated with E2 plus scrambled siRNA compared with their pre-transplantation tissues (2-induced adhesion of implanted endometrial fragments to the peritoneum of mice.To determine whether Ein siRNA A. Endomeantation A. The dimination B and C. mination D. To adj tissues A. In con tissues A. In the tissues B. These 2 is the most potent estrogen found in humans and plays a critical role in the development and progression of endometriosis (2 and β-catenin signaling in the pathogenesis of endometriosis is unknown. β-catenin is a major component of the canonical Wnt signaling pathway. In the absence of the Wnt ligand, this pathway is inactive due to continual degradation of non-junctional β-catenin. In the present study, we found that E2 increased β-catenin expression at the mRNA and protein levels in a time- and dose-dependent fashion in HESCs. Furthermore, E2 stimulated ESR1 binding to β-catenin. Taken together, our results showed that the β-catenin destruction complex failed to bind to the ESR1/β-catenin conjugation, leading to β-catenin stabilization and eventual translocation to the nucleus. The ESR1/β-catenin conjugation then associated with the transcription factors TCF3 and LEF1. When treated with ICI to antagonize the function of ESR1, β-catenin expression and stabilization decreased.E2 induced an increase in HESCs invasiveness and that ICI blocked this induced increase. β-catenin siRNA was then transfected into HESCs, which demonstrated that invasion was significantly decreased when β-catenin expression was inhibited. Similarly, VEGF and MMP9 gene expression were also inhibited. In the NOD–SCID mouse model, β-catenin siRNA abrogated the implantation of xenotransplanted endometrium mediated by E2, suggesting a novel and crucial mechanism in which E2 facilitates eutopic endometrial implantation through the β-catenin signaling pathway. However, the mechanism by which ESR1, β-catenin, and the LEF1/TCF3 complex regulate the expression of VEGF and MMP9 is unclear and requires further exploration.Adhesion, invasion, and angiogenesis are the steps in the fundamental pathological process of endometriosis. For ectopic implantation and growth to occur, endometrial tissue must first attach itself to the host tissue and then invade the host tissue and obtain its own blood supply from the local vasculature (2 in HESCs leads to the promotion of the nuclear translocation of β-catenin. We further found that ESR1, β-catenin, and LEF1/TCF3 interact in nuclei. All of these results suggested that E2 treatment leads to the accumulation of β-catenin in the nucleus, where β-catenin associates with the TCF/LEF family to activate the transcription of downstream target genes, such as the MMP and VEGF genes. These results confirm the importance of the β-catenin signaling pathway in endometriosis under conditions of abnormal estrogen levels.In the present study, we demonstrated that the molecular and functional activation of β-catenin signaling by E2 enhanced β-catenin expression and, as a consequence, β-catenin associated with the LEF/TCF family, increasing HESCs invasion and angiogenesis. In light of the multiple roles of the β-catenin signaling pathway in promoting the development of endometriosis, E2 may accelerate disease progression by up-regulating β-catenin expression. Therefore, these findings may provide a potential therapeutic target for the treatment of endometriosis.In summary, the present study demonstrated that E


Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9–39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through activation of GLP1R/Akt/GSK3b-mediated b-catenin signalling. In vitro studies have indicated that the induction of apoptosis in cardiomyocytes by palmitate is associated with the mitochondria-dependent apoptotic pathway (High levels of plasma free fatty acid seen in type 2 diabetes and obesity . ExcessiWnt and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signalling pathways converge through beta-catenin (b-catenin)-mediated regulation of cell survival/apoptosis in different cell types including cardiomyocytes . In respGlucagon-like peptide 1 (GLP1) is an incretin hormone synthesized and secreted by intestinal L-cells and stimulates insulin secretion from pancreatic b-cells in a glucose-dependent manner . NotablyGiven that b-catenin signalling plays a vital role in promoting cardiomyocyte survival and that GLP1 exerts direct cardioprotective effects upon lipotoxicity, we hypothesized that GLP1 could protect cardiomyocytes from palmitate-induced apoptosis via the activation of b-catenin signalling. To test this hypothesis, the present study was designed to investigate whether b-catenin signalling provides a mechanistic link in palmitate-induced apoptosis of isolated neonatal rat cardiomyocytes. Furthermore, the molecular mechanism underlying the protective effects of GLP1 on cardiomyocytes upon palmitate stress was characterized.Neonatal Sprague–Dawley rats (1–3 days old) were purchased from the Experimental Animal Centre of Guangdong Academy of Medical Science . Neonatal rat cardiomyocytes were isolated as previously described  pre-conjugated to BSA (Sigma–Aldrich) in 0.5% FBS–DMEM, in the absence or presence of GLP1  alone, or in combination of GLP1 with exendin-(9–39) (Sigma–Aldrich), a GLP1 receptor (GLP1R) antagonist or MK2206 , an Akt inhibitor, for up to 24 h. For drug interference, cardiomyocytes were pre-treated with the indicated reagents , MK2206) 1 h prior to palmitate treatment. The stock solution of 5 mM palmitate bound to 10% BSA was prepared using the methods described before (NM_053357) were designed using RNA structure 4.4 Software . Next, we generated the short-hairpin RNA (shRNA) against b-catenin through a DNA vector-based strategy. The pDC316-ZsGreen-sh-b-catenin vector was constructed by inserting annealed double-stranded oligo DNA with BamHI and EcoRI restriction enzyme sites  assay kit . The absorbance was measured at 450 nm using an automated microplate reader.To measure intracellular lipid accumulation, primary cardiomyocytes were stained by Oil red O dye (Sigma–Aldrich). After fixation using 4% paraformaldehyde, cells were washed and incubated with the Oil red O working solution (3 mg/ml) for 30 min at room temperature. Cells were then counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for highlighting nuclei, followed by imaging with an OLYMPUS FV1000 confocal laser-scanning microscope. Oil red O staining was detected under bright field and polarized light.A TUNEL assay was performed with a Cell Death Detection Kit  to detect the apoptosis of isolated cardiomyocytes. TUNEL-positive cells were counted in ten different microscopic fields of at least three independent experiments. The index of apoptosis was presented as the percentage of TUNEL-positive cells to total (DAPI-positive) cells using Image-Pro Plus Software .Neonatal rat cardiomyocytes cultured on glass coverslips were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. After blocking unspecific binding by incubating for 30 min with 1% BSA, cells were incubated overnight at 4 °C in the presence of a rabbit anti-active b-catenin (non-phosphorylated at Ser33/37/Thr41) antibody  or a rabbit anti-CD36 antibody . Cells were then incubated at 37 °C for 1 h with an Alexa-Fluor-546 or -488 labelled goat anti-rabbit  secondary antibody and then counterstained with DAPI. Images were obtained with an Olympus FV1000 confocal microscope.7 cells/dish were fractionated using the FractionPREP Cell Fractionation Kit . To detect the subcellular distribution of CD36, plasma membranes were extracted from 1×107 of cardiomyocytes using a sucrose cushion (38% wt/vol) as previously described  for 0–24 h. There was a dose- and time-dependent effect of palmitate on cardiomyocyte viability as determined by a CCK8 assay . Stimulab-Catenin is a transcriptional regulator of several genes involved in survival/anti-apoptosis. We thus investigated whether palmitate-induced cardiomyocyte apoptosis involved in inactivation of b-catenin signalling. Fluorescence images clearly showed that cells treated with palmitate resulted in markedly reduced cytosolic expression and almost lack of nuclear staining of active (non-phosphorylated) b-catenin, as compared with control . WesternGLP1 has been described to exhibit anti-apoptotic effects in a few models . CompareWe next determined whether GLP1 could restore the attenuated b-catenin signalling induced by palmitate. As shown in P<0.01) in Ad-sh-b-catenin-infected cardiomyocytes, compared with scramble control Ad-sh-sc  or shRNA for scramble sequences (Ad-sh-sc). The transduction efficiency with recombinant adenoviruses after 24 h was ∼95% as determined by the expression of ZsGreen in cardiomyocytes infected by Ad-sh-b-catenin or Ad-sh-sc. Infection with the adenovirus had no detectable effect on cardiomyocyte morphology and viability . WesternAd-sh-sc . We nextAd-sh-sc  upon lipAd-sh-sc . MoreoveAd-sh-sc . TogetheGLP1 acts through its cognate G-protein-coupled receptor, GLP1R, which is expressed in several tissues including the heart . To deteAkt activation leads to GSK3b phosphorylation (Ser9) and inactivation and thus the stabilization of b-catenin. We first investigate whether Akt is required for the beneficial effect of GLP1 on palmitate-treated cardiomyocytes. Cardiomyocytes were incubated with palmitate for 24 h in the absence or presence of GLP1 alone, or in combination of GLP1 with MK2206 . Compared to GLP1 alone, co-administration of MK2206 failed to suppress the palmitate-induced apoptosis of cardiomyocytes, which led to a significant increase in TUNEL-positive cells  and caspThe modulation of upstream signals of b-catenin in the cardiomyocytes was further investigated. Compared with the BSA control, palmitate significantly reduced the phosphorylation of Akt (Ser473) and increased GSK3B activation as demonstrated by the impairment of phosphorylated-Ser9–GSK3B expression, which led to a dramatically decreased stability of b-catenin as demonstrated by reduced expression of active b-catenin (non-phosphorylated at Ser33/37/Thr41) . As a reLast, we also assessed the effect of palmitate on intracellular lipid accumulation in cultured cardiomyocytes, as intramyocardial accumulation of triacylglycerol metabolites may contribute to diabetic/lipotoxic cardiomyopathy . As repoOur present findings first highlight the importance of GLP1-mediated b-catenin signalling in protecting cardiomyocytes from palmitate-induced apoptosis. To our knowledge, this study represents the first demonstration that b-catenin signalling was attenuated in a model of palmitate-induced cardiomyocyte apoptosis, which was restored by GLP1 treatment.in vitro . The GLP1R-dependent protective effect of GLP1 observed in our study was consistent with some previous studies  the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis; ii) a restoration of b-catenin signalling by GLP1 is sufficient to protect cardiomyocytes from palmitate-induced lipotoxicity; iii) the anti-apoptotic effects of GLP1 are dependent on downstream activation of b-catenin signalling and mediated by a GLP1R/Akt/GSK3b axis; iv) GLP1 can prevented palmitate-induced CD36 membrane translocation and intracellular lipid accumulation in cardiomyocytes via activation of Akt. Our findings suggest a potential therapeutic target for b-catenin signalling and also a prospective beneficial role for GLP1 in palmitate-induced cardiomyocyte apoptosis. These results may also provide implications in the treatment of diabetic/lipotoxic cardiomyopathy.in vitro experiments.Y Y conceived and designed the study, carried out the studies, performed data analyses and interpretation, and wrote the manuscript. H Z participated in study design, carried out the experiments, performed data collection, and analyses. Z L participated in research concept and critical reading of manuscript. X M provided critical comments on this study. S L participated in the


Male Wistar rats were fed with a normal diet and a high-fat and high-cholesterol diet with or without AEERB. After treatment, biochemical assays of serum, liver and feces lipid levels, the antioxidant enzyme activity, malondialdehyde (MDA) and protein carbonyl were determined. Result: AEERB is completely soluble in water and rich in hydrophilic and lipophilic functional ingredients. AEERB scavenged DPPH• and ABTS•+ and exhibited antioxidant activity slightly lower than that of ascorbic acid in the linoleic acid system. The administration of AEERB reduced serum lipid levels and the atherogenic index compared with those of the hyperlipidemic diet group (HD). The administration of AEERB significantly lowered liver lipid levels, inhibited hepatic 3-hydroxyl-3-methylglutaryl CoA reductase activity, and efficiently promoted the fecal excretion of total lipids and total cholesterol (TC) (p < 0.05). Dietary AEERB enhanced antioxidant status in the serum, liver and brain by increasing the antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and decreasing the content of MDA and protein carbonyl. Conclusions: The results indicated that AEERB might act as a potent hypolipidemic and antioxidant functional food.Purpose: The aqueous enzymatic extract from rice bran (AEERB) was rich in protein, γ-oryzanol and tocols. The aim of this study was to investigate the effects of AEERB on the regulation of lipid metabolism and the inhibition of oxidative damage. Methods: The antioxidant activity of AEERB Hyperlipidemia is a risk factor for the development of cardiovascular diseases (CVD), including atherosclerosis (AS), coronary heart disease (CHD), and hypertension. CVD is the major cause of human death in China as well as other nations in the world . NumerouRice is widely consumed globally, and rice bran (RB) is an important by-product of rice milling; RB contains the aleurone layer and some parts of the rice endosperm and germ. Recently, RB has attracted much attention because of its comprehensive nutritional and functional properties ,6,7,8. R in vitro and in vivo.Dietary change is the first treatment for improvement of metabolic syndrome symptoms ,15. We dThe RB was provided by the Great Northern Wilderness Agriculture Co., Ltd. . From Sigma-Aldrich Co., Ltd. , we obtained 2,2-diphenyl-1-picrylhydrazyl (DPPH), trypsin (1:250) and tocopherol and tocotrienol isomers, and 2,2ʹ-azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS) and γ-oryzanol were purchased from TCI ; 2,4,6-tripyridyl-s-triazine (TPTZ) was obtained from Fluka . Folin-Ciocalteau reagent was purchased from Solarbio Technology Co., Ltd. . Standard of rutin was obtained from NICPBP , and butylated hydroxytoluene (BHT) and ascorbic acid (AA) were purchased from Sinopharm Chemical Reagent Co., Ltd. . The other chemicals were of analytical grade.AEERB was prepared according to an enzymatic process previously reported by our laboratory with slight modifications . The RB The chemical composition of AEERB was analyzed as follows: the total protein content was determined by the Kjeldahl method (a nitrogen conversion factor of 5.95); the total fat content was determined by the Soxhlet method; the reduced sugar content was determined by the Somogyi method ; the tot•+ scavenging activity [There are many methods to determine the antioxidant capacity, and each one has its limitations. Different antioxidant assay methods might yield different activity trends because of the reaction mechanisms ,22,23. Tactivity ; (4) linactivity . The lipn = 10, each) and were given different dietary treatments. During the 42-day period, two control groups were fed a normal diet (ND) or a hyperlipidemic diet (HD) [The male Wistar rats  were conditioned to the environment of the animal house . The rats were allowed free access to commercial pellets  and tap water for 7 days before the experiment. After one week of acclimatization, the rats were randomly divided into three groups of similar average body weight (iet (HD)   as well as the SOD, CAT and GSH-Px activity and the MDA and protein carbonyl content in the serum, liver homogenates and brain homogenates were determined with enzymatic methods using corresponding commercial kits .The concentrations of TC, triglyceride (TG), LDL-C, HDL-C, ApoA, ApoB and Lp (a) in the serum lipids were measured using an Automatic Chemistry Analyzer . The extraction of the lipids from the liver and fecal was based on the method of Folch and others , and theThe hepatic HMG-CoA reductase activity was measured by ELISA methods with commercial kits .p < 0.05. The analysis was performed with the Statistix 8.0 software package .All the parameters were expressed as the mean ± SD. The results were statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison tests. The criterion for significance was The main components of AEERB and RB are presented in n-hexane extracts. In this study, the antioxidant activity of AEERB that was soluble in water was investigated by different principles. As summarized in •+ scavenging activity; however, AEERB showed DPPH• scavenging activity similar to that of BHT and inhibition lipid peroxidation similar to that of AA. Among these methods, two types of radical scavenging activity analyses were used to evaluate the antioxidant capability of antioxidants with a hydrogen donor. Because of the relatively high level of polyphenol and flavonoid compounds in AEERB, AEERB had radical scavenging activity and the IC50 values of DPPH• scavenging activity and ABTS•+ scavenging activity were relatively close. These results indicated that AEERB had a noticeable antioxidant activity in fat-soluble systems and in water-soluble systems. AEERB might serve as a natural and safe antioxidative functional food or nutraceutical.The antioxidant potential of RB had been documented; it was concentrated in certain bioactive compounds of alcohol extracts or The growth parameters of the rats are presented in p < 0.05), respectively. There was no significant difference in the serum GSH-Px, CAT and MDA content among the experimental groups. A similar trend of antioxidant status change was observed in the liver tissue and brain tissue (p < 0.05). The antioxidant effect in the brain tissue was not significantly enhanced in AEERB group (p > 0.05), compared with that in the HD group. The antioxidant enzyme levels and activities  in the brain tissue are relatively low and enzymes metabolize slowly, which leads to a difficult recovery of the cumulative effect of oxidative damage. The brain tissue MDA and protein carbonyl were not significantly different in all the treatment groups. Previous studies have shown that hyperlipidemia could destroy the antioxidant defense system; decrease the activity of SOD, GSH-Px, and CAT; and elevate the lipid peroxide level [2O2 by SOD and then detoxified by CAT [2O2 to protect the structure and the function of cell membranes from the damage and interference of peroxide. It is crucial to maintain enzyme activities to accommodate these oxidative stresses and reduce oxidative damage [As shown in n tissue . The antde level . Hyperchd by CAT . GSH-Px e damage . In thise damage . Our datp < 0.05, except ApoB), respectively, whereas HDL-C and ApoA increased by 32.76% (p < 0.05) and 22.73%, respectively, as compared with the HD group. The AEERB group showed more a notable effect in TC and TG than in the lipoproteins. No significant difference in ApoA and ApoB was observed between the HD group and the AEERB group (p > 0.05). Excessive LDL-C occurs in blood deposits in blood vessel walls and becomes a major component of AS plaque lesions. HDL-C facilitates the translocation of cholesterol from the peripheral tissues such as arterial wall tissue to the liver for catabolism [1 and AI2 atherogenic indexes express the risk extent of AS. A lower index value of index indicates a lower extent of AS. This study showed AI1 and AI2 were significantly reduced in the AEERB group compared to the index levels in the HD group (p < 0.05). Our data clearly indicated that administration of AEERB lowered cholesterol and reduced the risk of AS and CHD; AEERB might be used in the treatment of AS. Lp (a), an independent risk factor for CHD, is predominantly synthesized in the liver; its major function is to prevent the dissolution of blood clots and to promote the formation of AS. Angina pectoris, myocardial infarction, and cerebral hemorrhage are closely related to increased Lp (a) levels. In this experiment, the Lp (a) of the AEERB group was much lower than that of the HD group . The positive effect of AEERB on apolipoprotein suggested that apolipoprotein was regulated by AEERB to combine with a corresponding receptor as the ligand, take part in lipoprotein metabolism and return lipid metabolism to normal, thereby playing a role in regulating lipid levels and lipoprotein metabolism disorders.This study explored the effect of AEERB on serum lipids in hyperlipidemia rats. tabolism . DecreasIn addition, combined with the results of the antioxidant experiments, it was clear that serum TC, TG, and the atherogenic index were negatively correlated with the antioxidant indicators  and positively correlated with the MDA and protein carbonyl content. The lipid levels in the hyperlipidemic rats and their oxidation-antioxidant levels were shown to be closely related and mutually promotive. AEERB showed consistency in its lipid-lowering and antioxidative effects and had significant physiological lipid-lowering and antioxidative activities.p < 0.05), whereas the consumption AEERB showed an effect on reducing the lipid levels. Except for the free cholesterol, the lipid parameters of the AEERB group were obviously lower than those of the HD group (p < 0.05). This result was similar to that of a previous study regarding the effect of RB oil on the regulation of liver TC and TG [The liver lipid profile exhibited a similar tendency as that of the serum lipid profile. As shown in C and TG ,36. TemeC and TG  reportedC and TG . The lowp < 0.05). There was no significant difference in the fecal excretion and TG content in the experimental groups (p > 0.05). The fecal TC was considered another marker for the metabolism of cholesterol. In the intestinal mucosa, phytosterols, particularly sitosterol, could compete with and inhibit cholesterol absorption. There was high level of γ-oryzanol in AEERB. Thus, we hypothesize that the increase in the fecal lipid levels in the AEERB group might be because of the inhibition of cholesterol absorption in the intestinal tract.The fecal excretion and lipid levels are shown in The liver is the main organ of endogenous cholesterol biosynthesis, and HMG-CoA reductase is the key rate-limiting enzyme in the biosynthesis pathway of isoprenoids and cholesterol. We measured the activity of HMG-CoA reductase to determine whether the decrease in the liver cholesterol level was because of the inhibitory effect of this enzyme . The datThe pathological examinations of liver sections of various groups are shown in Cholesterol homeostasis is precisely controlled by the regulation of endogenous cholesterol synthesis and the absorption of exogenous cholesterol. We concluded that the hypolipidemic mechanisms of AEERB could be elucidated as follows: first, AEERB inhibits the absorption of cholesterol (phytosterols and γ-oryzanol) and enhances their excretion in feces; second, AEERB effectively reduces endogenous biosynthesis (tocols) by inhibiting the hepatic HMG-CoA reductase activity. The precise hypolipidemic mechanisms remain for detailed investigation in the future because this study did not include research on the excretion of bile acids and the relative expression of cholesterol and lipoprotein metabolism-related genes in the hepatic mRNA. This study suggests that AEERB, given its antioxidant and hypolipidemic activity, could be an excellent source for functional foods or nutraceuticals.


IL10 expression.In the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and To investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out.Flow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS.in vitro LPS treatment, Western blot analysis demonstrated an active cross talk between canonical Wnt signaling and NF-кB pathway that is essential to negatively control enteric neuronal response to inflammatory stimuli. Upon costimulation with LPS and Wnt3a, a significant anti-inflammatory activity was detected by RT-PCR based on an increased IL10 expression and a downregulation of pro-inflammatory cytokines TNFa, IL1B, and interleukin 6 (IL6). When the availability of neurotrophic factors in ENS cultures was abolished, a changed cell reactivity by Wnt signaling was observed at basal conditions and after LPS treatment.Under The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS. Moreover, under active inhibition induced by GSK3β or cross talked pathways , deactivate GSK3β [p(Ser9)-GSK3β], and tag β-catenin for proteosomal degradation [p(Ser33)-β-catenin]. In order to evaluate a cross talk between Wnt and NF-кB pathways, a protein expression analysis by WB was carried out to evaluate the phosphorylated state of Akt, GSK3β, β-catenin, and the nuclear translocation of both β-catenin (n-β-catenin) and NF-кB   , NGF, and LIF was observed in all samples without any significant modulation by culture conditions and stimulation. These evidences highlighted that ENSc might modulate their reactivity to exogenous Wnt3a and LPS at longer time of culture (T7) through an endogenous production of growth factors. For this reason, in our opinion, the specific effect of Wnt3a and LPS could be really discriminated at early time of stimulation (T1). As AXIN2, CJUN and CMYC are reported as target genes of β-catenin transcriptional activity, their expression (p ≤ 0.01) at T1  the gene expression of FZD9 and WNT3A (p ≤ 0.01) while decreased (p ≤ 0.05) the mRNA level of TLR4. Specific LPS-mediated response showed an increased expression (p ≤ 0.05) of TNFa, IL6, and IL1B genes while a reduced level (p ≤ 0.05) of IL10 mRNA was observed. The costimulation with LPS and Wnt3a reversed the expression pattern of ENSc (p ≤ 0.01). Moreover, Wnt3a demonstrated to exert a negative control (p ≤ 0.01) on the gene expression of pro-inflammatory cytokines and to enhance ENS defense promoting an increased expression of anti-inflammatory IL10 (p ≤ 0.01) and FZD9 (p ≤ 0.01). Probably due to a feedback loop aimed to restore homeostatic conditions, TLR4 expression was increased by Wnt3a (p ≤ 0.05) . A fine control of these processes guarantees that a sufficient number of NCCs enter the foregut at the correct time while proliferation and differentiation are maintained at a balanced level at migration wavefront. It is known that the microenvironment plays a pivotal role to regulate the extent of ENS formation influencing on NCC number and gut colonization. Although neurotrophic factors are demonstrated to be essential for gut formation, the biological processes controlled by these factors are not yet completely elucidated ,95. In bl marker , it has l marker ,100, putl marker , and mesl marker . In the l marker , and ENSl marker  that, lil marker ,105-108,l marker ,105 and l marker ,109. In l marker , the expin vitro maintained and numerically increased when deprived of bFGF, EGF, GDNF, and NGF. These evidences suggested that a diminished availability of neurotrophic factors fostered neuronal commitment of progenitors but negatively controlled the cell differentiation process as shown by lacking neurosphere formation. In contrast, under standard culture conditions, glial differentiation was promoted more than neuronal and neurospheres were regularly formed.As suggested by the expression level of Sox10, p75, and GFAP, glial cells were stimulated under standard culture conditions while neuronal committed cells expressing Sox2, NG2 ,111, andUnder Wnt3a (20 ng/mL) and LPS (5 μg/mL) stimulation, neurospheres were demonstrated to increase in number and size. After 1 day of culture, the neurosphere diameter was 10 μm bigger in LPS- and Wnt3a-treated samples than that observed in control. At T7, the number of neurospheres was doubled in LPS-treated samples compared to that detected under Wnt3a stimulation. Interestingly, a larger diameter of neurospheres was induced by Wnt3a with respect to LPS. The different proliferation effect could be indicative of a protective mechanism that, through the stimulation of immature cells but by the activation of different pathways, LPS  and Wnt3in vitro rat model.Although FZD9 in human has been shown to bind Drosophila Wingless , the firThe interaction between Wnts and Frizzled receptors is specific and differently involved in physiological and pathological conditions ,113. In Wnt1 and Wnt3a are coexpressed at the dorsal midline of the developing neural tube and control the midbrain patterning and the formation of the paraxial mesoderm, respectively. When a deficiency of both Wnt1 and Wnt3a is observed, the development of neural crest derivatives is compromised suggesting that local Wnt signaling regulates the expansion of dorsal neural precursors. Despite the high number of mammalian Wnt and Frizzled members, a poor characterization of Wnt Frizzled binding specificities is reported.ex vivo cultured neuronal population. As previously reported [In particular, it is known that canonical Wnt signaling controls crypt progenitor gene expression pattern . Recentlreported , the regreported  and neurreported . In our reported . The expreported . In the The cross regulation of Wnt and NF-кB signaling pathways has been shown to modify the biological effects of gene expression during development, immune function, inflammation, and carcinogenesis. A wide spectrum of phosphorylation/acetylation events and associating proteins is demonstrated to modulate the activation or inhibition of β-catenin, NF-кB, or their binding activity to DNA , indepenIn the present work, we have demonstrated that canonical Wnt signaling is active in ENS and cross talks with NF-кB modulating the response to LPS. Phosphorylation events at serine 473 activate Akt and lock it at active conformation . In bothin vitro model is not based on the inhibition of PI3K/Akt signaling pathway but only on the deprivation of main ENS trophic factors. This activity is hypothesized to be specifically dependent on the activation of Wnt and NF-кB signaling, probably modulated by GSK3β but not depending on exogenous growth factors such as bFGF and EGF as the evidences were taken from basal medium-treated samples. The concomitant presence of p(Ser33)-β-catenin and p(Ser9) GSK3β at early phase of Wnt3a and LPS stimulation suggested that alternative mechanisms of phosphorylation might occur promoting the negative control of β-catenin and thus its modulation. As the protein expression profile detected in standard and basal medium-treated samples showed a stronger accumulation of β-catenin at late phase of Wnt3a stimulation, GSK3β downregulation by simultaneous treatment with Wnt and growth factors is hypothesized to involve GSK3β pools differently modulated by Akt.As p(Ser9)-GSK3β was detected only in standard medium and p(Ser33)-β-catenin started to be expressed at T1h, we hypothesized that Akt controls phosphorylation events in resting cells promoting directly or indirectly both activation and degradation of β-catenin, besides nuclear translocation of NF-кB p65. It was demonstrated that Akt phosphorylates β-catenin at Ser552 leading first to its dissociation from cell-cell contacts and then to the increase of its binding to 14 3 3 proteins and its transcriptional activity . As GSK3in vitro studies on neuronal cell lines or primary cultures. Phosphorylation of Akt through phosphatidylinositol 3 kinase is essential to promote ENS precursor survival [in vitro model could simulate pathological conditions, wherein the limited or absent supply of growth factors promotes gut inflammation, the higher nuclear translocation of β-catenin could be interpreted as a negative regulation of NF-кB p65 transcriptional activity.The critical role of Akt signaling for neuroprotection against deprivation of growth factors , oxidatisurvival . Akt levsurvival . Interessurvival . As BM-bAs GSK3β has been shown to regulate NF-кB at the level of transcriptional complex  and β-caIn the present study, using Co-IP technique, we demonstrated that nuclear β-catenin and NF-кB p65 could complex together or are present as free proteins in resting ENS cells. The β-catenin/p65 complex and the free proteins resulted to be differently expressed in dependence on experimental conditions. It is already reported that β-catenin can physically interact with p65 and p50 subunits to inhibit the binding of NF-кB to DNA in IкBα-independent manner and in association with additional cellular factors . InteresThe major expression of β-catenin/NF-кB p65 complex in SM-treated samples was correlated to the greater accumulation of nuclear β-catenin and NF-кB p65 deriving from aspecific stimulation of NF-kB  and Wnt signaling pathway exerted by trophic factors . Only thThe specific responsiveness of ENS cells to Wnt3a or LPS stimulation was confirmed evaluating the transcriptional activity of nuclear β-catenin and p65 subunit in samples cultured in basal medium. Both canonical Wnt signaling and NF-кB pathway are expressed in ENS and control gut homeostasis . As the TLR4 mRNA, suggesting a possible negative feedback scenario in which a positive regulation of Wnt pathway could be exerted by NF-кB signaling to restore homeostatic conditions. This hypothesis was further confirmed evaluating that the specific LPS-mediated inflammatory response was reversed in the presence of Wnt3a as demonstrated by a reduced expression of TNFa, IL6, and IL1B genes and, interestingly, an increased level of IL10 mRNA. As it is reported that LPS-treated enteric neurons respond stimulating the secretion of inflammatory cytokines such as IL6, TNFa [Schaale et al.  describeL6, TNFa  to activIL10, TLR4, and FZD9 expression. In turn, LPS balances the inflammatory response stimulating Wnt signaling and enhancing the transcription level of FZD9 and Wnt3a.Taken together these considerations, we concluded that Wnt3a exerts a negative control of NF-кB transcriptional activity in rat myenteric plexus and contributes to ENS defense by the stimulation of in vitro-simulated inflammation, Wnt signaling was demonstrated to exert an anti-inflammatory activity to negatively control NF-кB pathway.The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. As detected in freshly isolated ENS cells, FZD9 was hypothesized to be expressed in constitutive manner for some homeostatic activities. Although the total number of FZD9 positive cells cultured under standard and basal conditions did not significantly change, the expression level of FZD9 was upregulated in response to GDNF, bFGF, and NGF, suggesting a possible involvement of Wnt signaling in neuronal and glial differentiation. Interestingly, under


The two compounds inhibited Wnt signaling pathway in a concentration-dependent manner and exhibited selective cytotoxicity toward a number of colon carcinoma cell lines SW480, HCT116, and HT29, with only weak cytotoxicity towards the normal colonic epithelial cell line CCD-841-CoN. Rabdoternin B and maoecrystal I treatment induced G2/M phase arrest efficiently in SW480 cells as revealed by flow cytometry analysis. A further study found that maoecrystal I decreased the expression of Wnt signaling target genes, including c-myc, cyclin D1, survivin and Axin2 in colon cancer cells. Collectively our data suggests that rabdoternin B and maoecrystal I are novel inhibitors of canonical Wnt signaling pathway and may possess potentials for colon cancer therapy.Constitutively active Wnt signaling frequently occurs in most colon cancers. Therefore, inhibitors of Wnt signaling pathway could provide rational therapeutic effects for colorectal malignancy. Within this paper, we identified two inhibitors of Wnt signaling pathway, rabdoternin B and maoecrystal I from a natural  When ceAPC, the tumor suppressor gene, mutated in about 80 % colon cancers [β-catenin, Axin2 and GSK-3β that lead to hyperactive Wnt signaling are also found in colon cancers [Colon cancer is a common disease and is one of the leading causes of cancer death . APC  is widely distributed plants, many of which are used for the treatment of cancer and inflammation in traditional Chinese medicine. Over the past twenty years, the structures and bioactivities of their diterpenoids constituents, especially those with an ent-kaurane skeleton, have received considerable phytochemical and biological attention [ent-kaurane diterpenoids with dual-luciferase reporter gene assay [ent-kauranoids isolated from the aerial parts of Isodon rosthornii [Isodon xerophilus [Natural products and their derivates, with their structural diversity, offer a rich resource for identification of novel inhibitors of Wnt signaling. The genus ttention . In the ne assay  and idensthornii  and leavrophilus , respectent-kauranoids from a natural compounds library were screened using HEK293 cells that were stably co-transfected with Topflash, renilla reporters and wnt3A, namely HEK293W cells [50) of these primary hits on Wnt signaling pathway, we performed secondary screening using three concentrations by two-fold dilution, with the highest concentration being 20 μM. As shown in Fig. 50 values of 13.95 ± 0.66 µM and 10.58 ± 0.82 µM, respectively.To identify compounds that inhibit the canonical Wnt signaling pathway, two hundred 3W cells . CompounAPC mutated [β-catenin on site S45 [It is well known that Wnt signaling plays a critical role in regulation of cell growth, proliferation, and survival. Constitutively active Wnt signaling are found to be involved in about 90 % colon cancer cells . Moreove mutated , and HCTsite S45 , which aAs Wnt signaling plays an important role in the regulation of cell cycle, we measured the effects of rabdoternin B and maoecrystal I on the cell cycle of colon cancer cells. As shown in Fig. Axin2 in SW480 cells and knockout of Axin2 in mouse embryo fibroblasts (MEF) cells both enhanced the fraction of cells at the G2/M phase. Moreover, there is a hypothesis that Axin2, as a signaling, modulates the mitotic spindle checkpoint [Several studies demonstrated that Wnt signaling influences cell cycle by the up-regulation of G1 effectors on both a transcriptional and translational level . Howevereckpoint . Our resC-myc, cyclin D1, survivin, and Axin2 are well-known target genes of Wnt signaling pathway [c-myc, cyclin D1, survivin, and Axin2 in a dose-dependent manner, which showed that the inhibition of cancer cell growth is associated with Wnt signaling inhibition by maoecrystal I.Since rabdoternin B and maoecrystal I were identified to be inhibitors of Wnt signaling, we wanted to identify if the inhibition of cancer cell growth by the two compounds is due to the inhibition of Wnt signaling. Compared with rabdoternin B, maoecrystal I exhibited more potent cytotoxic effects and more remarkable cell cycle arrest activity in colon cancer cells, therefore, we explored the effects of maoecrystal I on endogenous Wnt signaling pathway in colorectal cancer cells.  pathway –26. The Regulation of β-catenin level is a feature of Wnt signaling pathway. Wnt/β-catenin signaling is activated after β-catenin accumulates and translocates to the nucleus and binds to other transcription factors . To inveRegulation of β-catenin to inhibit Wnt signaling pathway usually occurs on two distinctive levels, promoting β-catenin degradation or inhibiting association of β-catenin and transcriptional factors. Our findings reveal that maoecrystal I did not affect total β-catenin at the protein level, suggesting that maoecrystal I has no influence on β-catenin destruction. Therefore, we deduce that maoecrystal I may inhibit Wnt signaling pathway downstream of β-catenin destruction, such as suppressing the translocation of β-catenin into nucleus or reducing the association of β-catenin with transcription factors TCF4/LEF1, two important events that are downstream of β-catenin degradation.ent-kauranoids, rabdoternin B and maoecrystal I, as novel inhibitors of Wnt signaling pathway were identified in our study. Both rabdoternin B and maoecrystal I inhibited the growth of colon carcinoma cells and arrested SW480 cells at G2/M phase, which were associated with decreased expression of Wnt target genes. Maoecrystal I did not affect β-catenin level in SW480 cells, suggesting that maoecrystal I might inhibit Wnt signaling though downstream events of β-catenin degradation. In culmination, rabdoternin B and maoecrystal I, as novel inhibitors of canonical Wnt signaling pathway, may possess the potentials for colon cancer therapy.Two natural Isodon rosthornii [Isodon xerophilus [SW480, HCT116, HT29, and CCD-841-CoN were obtained from ATCC. RPMI 1640, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotics–antimycotics solution and trypsin–EDTA were purchased from HyClone . Mouse monoclonal anti-β-catenin antibody was from BD; Mouse monoclonal anti-Cyclin D1, anti-survivin, anti-β-actin antibodies were from Santa Cruz Biotechnology; Rabbit multiclonal anti-phospho-β-catenin (S33/37/T41), mouse monoclonal anti-GSK-3β, anti-phospho-GSK-3β (S9), rabbit anti-Axin2 antibodies were from Cell Signaling Technology; Hygromycin B, G418 and EGCG were purchased from Sigma. Rabdoternin B and maoecrystal I were isolated from the aerial parts of sthornii  and leav2 in a humidified incubator at 37 °C.SW480, HCT116, and CCD-841-CoN were propagated in Dulbecco’s modified Eagle’s medium (DMEM), and HT29 were maintained in modified RMPI-1640 medium, both supplemented with 10 % fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin. HEK293W were cultured in DMEM medium, supplemented with 10 % fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL G418 and 100 μg/mL Hygromycin B. Cells were maintained with 5 % CO490 nm value) was measured using microplate reader (Bio-Rad). Cell viability was determined and the 50 % inhibitory concentrations (IC50) were calculated by Reed and Muench method.100 μL cells were seeded in 96-well plate with a density of 5000 cells per well the day before compounds treatment. Cells were incubated with test compounds with the concentration range 0.064–40 μΜ for 48 h in triplicates. Then cell viability was evaluated using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer’s instructions. The absorbency of each well (OD50) were calculated by Reed and Muench method.A stable transfected human kidney epithelial cell line HEK293W was created as described and used for assay of inhibitory effect of compounds on Wnt signaling . 100 μL SW480 cells were plated in 6-well plates and incubated overnight at 37 °C. Rabdoternin B and maoecrystal I were added, respectively, and the cells were incubated for further 48 h. Cells were then collected and fixed in pre-cooled 70 % ethanol at −20 °C for 1 h. After washing with PBS, RNase (25 μg/mL) was added for 30 min at 37 °C for digestion, and then stained with PI (100 μg/mL) in dark for 20 min. Fluorescence intensity was analyzed by FACS Calibur flow cytometer . The percentages of the cells distributed in different phases of the cell cycle were determined using ModFIT LT 2.0.Cells were seeded in 6-well plates and incubated at 37 °C overnight. Total cell lysates were prepared by direct lysis on ice in lysis buffer  after compounds treatment. Samples were then fractionated on 10 % acrylamide gel, transferred to a PVDF membrane (Bio-Rad), and incubated with specific primary antibodies followed by the corresponding peroxidase-conjugated secondary antibodies. Proteins of interest were visualized by chemiluminescent detection on an ImageQuant LAS mini4000 .t test using Excel 2007 software. In all the assays, the probability value (p) of less than 0.05 was considered as statistically significant.Statistical analysis was performed via paired-


The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders  have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need. The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance and a significant threat to human health , 2. YearAt the same time, it is becoming more and more difficult and expensive to develop new antibiotics as an adequate response to the phenomenon of multidrug resistance. Actually, very few new antibiotics are in the pipeline of the pharmaceutical industry at the moment .Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Bacteriophages (or “phages”) are viruses that infect and replicate within specific bacteria without harming others. Although bacteriophage therapy (hereafter BPT) was developed and practiced in Europe and the former Soviet republics, the western world abandoned the use. This was mainly because at that time (1930s) there was a lack of knowledge of what a bacteriophage really was (a virus) as well as the discovery of antibiotics. These molecules are well-characterized chemical substances, relatively easy to produce in a well-controlled fashion, initiating the golden age of antibiotics, the so called “miracle drugs” .Given the worldwide antibiotic crisis, the existing and continued experiences build up in Eastern Europe and the former Soviet Republics combined with recent encouraging animal and human study results; there is a growing interest for BPT in modern medicine and the agrobioindustry, a recognized potential reservoir for antibiotic resistant germs , 3, 7–10 four main obstacles. First, historical clinical data about the safety and effectiveness of BPT are not considered proven and validated by European regulators. Second, given the substantial costs and investment in the development and marketing of conventional medicinal products by the pharmaceutical industry, there is in our actual pharmacoeconomic model an imperative demand for a strong intellectual property (IP) protection. For now, such protection is rather fragile for natural lytic phages. Third, an efficient and effective BPT-concept needs to be flexible and tailored to the patient [Despite this growing interest, introducing BPT in the western medical world remains highly problematic as a consequence of patient , 12. Tha patient , 12. Rat patient . In view patient . E. coli 0104 outbreak as an example [ Escherichia coli caused a serious outbreak of food borne haemolytic uremic syndrome and bloody diarrhoea in Germany. Antibiotics were of questionable use and 54 deaths occurred, beside tens of clinical cases with lasting sequels [ E. coli O104:H4 outbreak strain [Recently, Henein emphasized that so far no bioethical bacteriophage therapy debate has been published. He gave the most recent example . Indeed  sequels , 17. Sevk strain –20. The Międzybrodzki et al.  addresseThe main purpose of this paper is not so much to fleece out these four main obstacles, nor to determine how an adequate regulatory framework for BPT might look like. This has already been done in other publications , 14, 22.Given the above nonnormative specifications of BPT, the basic moral problem associated with BPT can be formulated as follows. BPT, when used in a flexible  and sustainable manner, has the potential of saving thousands of lives every year , 24. How principle of nonmaleficence, which implies the obligation not to inflict harm on another. This principle is designed to protect the patient. The second principle is the principle of beneficence, which implies the obligation to prevent or to remove harm or the obligation to promote good [Central to this moral problem are the preservation and restoration of the health and well-being of the patient. Two basic underlying moral principles are relevant in this patient-centred approach and will be further investigated in this paper. The first is theote good . principle of nonmaleficence in the context of BPT. Indeed, a patient has a right not to be subjected to a medical treatment or therapy that has not yet been rigorously tested for its effectiveness and possible health risks (as is BPT). The moral duty, however, not to subject a patient to a not yet approved therapy is not an absolute, but a prima facie duty. This means that under well-defined, specific circumstances this duty not to subject someone to a not yet approved therapy can be set aside [Ethically Justified Medical Therapy” (EJMT). This is a medical therapy that has not yet obtained an official approval for its health effectiveness (at least not according to western standards), and/or for which some doubts remain concerning possible health risks, but the use of which seems to be morally acceptable given the specific circumstances of the case at hand. For such a therapy to be labelled as an EJMT, six criteria need to be met  may play a certain role as well. Therefore, if indeed a patient's health is at stake (a good reason), then our intention for using the yet unapproved therapy has to be about improving the patient's health condition and not about commercial, research, or cost-reducing benefits.Secondly, we need to make sure that all the moral agents involved are motivated by reasonable chance that the use of the therapy in a particular case will have the desired result. In case of BPT, based on preliminary examinations and testing by experienced specialists, multiple case studies reporting success were published [Next, there needs to be aublished –30.proportionality). It is important in this respect to try to avoid as much as possible undesired side effects related to BPT [Efficacy of bacteriophage therapy has not yet been proven according to European regulators, but anyway the concept of reasonable chance does not imply real proof of such efficacy. What is more, there must be a good prospect that the probable health benefits will outweigh the risks of subjecting the patient to the therapy  [Finally, a decision to subject a patient to a not yet approved therapy needs to be made in respect with the patient's right to autonomy (thority) . However prima facie duty. In specific circumstances (determined by the above criteria) war can be a morally acceptable option (for instance in case of self-defense or humanitarian intervention). A very similar moral mechanism is at work in our discussion about BPT. Subjecting a patient to a not yet approved therapy is morally wrong, but sometimes, in specific  circumstances (determined by the six criteria), it can be a morally acceptable option to subject a patient to such unapproved therapy, and not just a morally excusable option.Although these six criteria were originally developed within another context, namely, that of the just war theory (JWT), it seems that these criteria can also be of valuable use in the ethical discussion on BPT . This is case-by-case basis. Numerous types of bacterial infections exist. Any manner to combat such infection needs to be considered separately. In fact, medical ethical committees provide also case-by-case reasoning while evaluating proposals for treatment.Whether or not BPT is an EJMT needs to be checked on a precautionary regulatory framework. Until it can be adequately shown that BPT can live up to western health care standards, there will be a constant need to justify the use of BPT on a case-by-case basis. Moreover, given the specific nature of BPT (its interactive and ever-evolving character) and the need to focus on tailor-made solutions, it will be difficult, if not impossible, to claim once and for all  that BPT is effective and safe. This would also imply that any regulatory framework designed to assure the safety and effectiveness of flexible and sustainable BPT will never completely lose its precautionary character.We would like to conclude the first part of our argument by saying that the six criteria we used to evaluate BPT's moral permissibility form the ethical basis for some kind of morally permissible in that specific case. But showing moral permissibility for BPT is only a necessary condition for our purpose, which is demonstrating that it is simply morally unacceptable to obstruct  the accessibility to BPT. We also need to show that somehow there exists a moral duty to take the necessary steps in order to make flexible and sustainable BPT available in a more organized fashion.Demonstrating that BPT constitutes an EJMT in a sufficient number of cases is of course but a first part of our moral argument. Showing that in a specific case BPT is an EJMT makes its usePharmaceutical companies are the primary actors in the business of developing new and improved medicinal products and therapies. Hospitals do not have the financial capacity and resources to fully develop bacteriophage-based products according to the current pharmaceutical medicinal product guidelines. Although our initial focus will be on pharmaceutical companies, this does not mean that other actors (like public authorities) are absolutely absolved of all responsibility in this respect. We will come back to this specific issue later on in this paper. nonmaleficence provides a so-called prima facie moral protection for the patient against therapies and treatments that have not yet proven their health effectiveness and/or for which some doubts remain concerning their possible health risks. In trying to establish a moral duty to contribute to the development of lifesaving therapies, such as BPT [ beneficence.In our patient-centred approach, we perceived that the principle ofh as BPT , 30, 38,Despite the fact that it is not in the interest of the classic pharmaceutical industry, organized in accordance with the actual pharmacoeconomic environment, to invest in sustainable BPT (see the above mentioned obstacles), the beneficence-principle seems to provide a sufficient moral basis for arguing that the pharmaceutical industry has a duty to do so anyway.medicine is a moral enterprise whether it is practiced in the system of slavery or market economy” [In the 1970s, most supporters of market economy embraced Friedman's view that the social responsibility of business is to increase its profits, not to relax the conditions of profit-maximization on behalf of the wider interests of society . But, iseconomy” . Defendeeconomy” , 41.One of the reasons why the above conclusion is not that straightforward has to do with the specific nature of the beneficence-principle. Indeed this basic principle is especially morally relevant within the relation between the health care professional and his patient. Because of his specific role, it can be said that the health care professional has the moral obligation to undertake all the necessary and reasonable measures to improve his patient's health condition . The relational context between a patient and the pharmaceutical industry is of course very different from the one between the health care professional and his patient. It is no longer a relation of care, compassion, and beneficence, but a relation of an economic or a commercial kind. The industry's role is to develop, produce, and sell medicinal products, whereas the role of the patient is that of a consumer. This does not mean that the pharmaceutical industry has absolutely no obligations whatsoever towards its patient-consumers. The industry has the obligation to take all the necessary and reasonable measures to ensure the effectiveness and the safety of the medicinal products it decides to develop, produce, and sell . Within  utilitarian kind. One might quite convincingly argue that economic actors in our society, like private businesses and companies, do not only have specific client-related obligations, but also a somewhat broader social responsibility to promote overall well-being in society. If we agree that this is the case (that there exists such a utilitarian-based responsibility), then it still remains to be seen of course whether the moral requirement to promote novel antimicrobial approaches like BPT can indeed be based on such a notion of social responsibility. To verify this, we need to explore two separate questions.Perhaps a more promising line of argument is of a first question goes as follows: to what extent may we assume that the promotion of BPT  constitutes indeed the course of action that, compared to other possible alternatives, will lead to better results in confronting the health challenges related to an increasing antibiotic resistance problem in bacteria (hereafter the resistance problem)? Proving that BPT is the best option in tackling this specific health problem from a utilitarian point of view is essentially a technical matter that requires further thorough knowledge in microbiology and in health care economics. In other publications it has been shown that this may be the case [Thethe case , 43, 44. always promote the overall good. The key word here is “always”.Establishing with a reasonable degree of certainty that BPT is indeed a good option in confronting the resistance problem from a utilitarian point of view constitutes, however, a necessary, but not a sufficient condition for our purpose to demonstrate that there is such a thing as a moral requirement to promote BPT. A part of our common-sense morality clearly states that promoting the best overall result does not automatically generate a moral requirement to do so. We can illustrate this with an example. Suppose that ten people are on the verge of losing their lives (that they are about to drown). Suppose also that person A could save all of them if he wanted to. The only trouble is that person A can only do so with a considerable risk to himself. Although, saving the ten lives (even with the risk of losing his own life in the process) is the best option from a utilitarian point of view; there is a general agreement within common-sense morality that person A is under no moral obligation to do so. In this specific example person A is allowed to favour his own life, even if sacrificing it in order to save the ten others would be preferable from a utilitarian viewpoint. In the theory of normative ethics, common-sense morality contains so-called agent-favouring options (or agent-favouring prerogatives) . These a obligatory act, but rather a supererogatory act inspired by idealism [Indeed, in some cases the promotion of the overall good does create a moral requirement to do so. For instance: if person A could save the lives of those ten people with practically no risk for himself, he can no longer avoid the obligation to do so. In order to understand the mechanism of the agent-favouring options better, it is essential to take into account two factors: the (probable) cost to the agent, and the (probable) amount of overall good or well-being that is at stake. If the amount of good at stake outweighs the cost to the agent (who has no risk to lose his life), then the requirement to promote the overall good can no longer be blocked by the agent-favouring option. This seems to suggest that the option protecting a moral agent's self-interest is characterized by some kind of threshold, the level of which is determined by the cost the moral agent will have to pay when he decides to serve the overall good. Once the amount of good or well-being at stake crosses that threshold, the creation of a moral requirement to bring about this amount of good can no longer be blocked by the agent-favouring option. A general rule seems to be then: the higher the cost to the agent, the higher the threshold, the stronger the agent-favouring option, and the lesser the probability that the agent will be subjected to the obligation to promote the overall good. It should be clear, however, that whenever the interests of an agent are protected by such an agent-favouring option, he or she is still at liberty to promote the overall good. If, for instance, person A is a heroic kind of a person and he will not hesitate to save those ten people, even if this means sacrificing his own life. It is obvious that such an act will not be morally condemned. Far from it: such an act will typically be praised and admired. But again, it is not anidealism .Let us assume for the sake of argument that such agent-favouring options are indeed a part of our common-sense morality. This being the case, it might very well be that the economic and commercial interests of the pharmaceutical industry are going to be protected by such options. Even if it would appear that promoting BPT is indeed the best choice in tackling the resistance problem, pharmaceutical enterprises cannot be morally obligated to sacrifice their own commercial interests in order to give priority to the development of BPT. It is possible, however, that some pharmaceutical companies could decide to go ahead anyway with the development of BPT. But, again, such a decision would constitute a supererogatory act, not a mandatory act. second question: is it reasonable to assume that there exists such an agent-favouring option that protects the private interests of the pharmaceutical industry and thereby blocks the creation of a moral requirement to promote BPT? Answering this question requires a more profound cost/benefit analysis. In case of pharmaceutical companies' investments in bacteriophage product development, costs are considerable since all regulatory processes need to be conducted [ moral force of that option and will it be strong enough to block a moral requirement to promote BPT? At this point in the argument we will need to evaluate the importance of the cost to the pharmaceutical industry. This will indeed give us some idea of the moral force of the agent-favouring option . Today, any organization, pharmaceutical company, or any nonprofit actor like a hospital, willing to implement BPT, is expected to follow the classical marketing authorization and market placement procedures. The costs for developing BPT following these frameworks can be as much as 400–800 million USD [We now come to ouronducted . Assuminlion USD  and not In addition, intellectual property rights (IPRs) that in general provide owners exclusivities to recoup investment costs are hardly unavailable (or weak) in the context of BPT . The reaWill the overall health benefits of saving thousands of lives, generated by the introduction of BPT, cross the option's threshold ? To verify this, we need to compare the costs of investment in BPT development by pharmaceutical companies with the unproven potential BPT of, for example, savings of the lives of 25.000 Europeans each year , 30, 38,According to the authors, previous analysis demonstrates that arguments can be found to support a moral requirement for the pharmaceutical industry to promote BPT. It remains to be seen what specific form this moral requirement will take. The pharmaceutical industry, due to its specific knowledge and know-how, will certainly have some role to play in the validation process of the clinical data relating to BPT (obstacle (1)) and/or in furthering the research concerning the potential negative coevolutionary consequences of unlimited use of BFT (obstacle (4)). In addition, it is important to consider how in the future industrial companies will maintain (keep on respecting) this moral duty and how these companies can be made aware of the importance of creating and maintaining high moral standards in the long run. History portrays similar developments, more specific in the domain of cell and gene therapy. Several regulatory incentives, specific types of subsidies and models for public-private partnerships have contributed to stimulate the industry for investments into these therapies . More inUntil now we have solely focused on the pharmaceutical industry as the principal bearer of this potential moral requirement to promote BPT. Obviously, political authorities, much more so than private companies, have a social responsibility to promote public health in the most efficient way they can. What is more, due to their public nature, public authorities cannot hide behind cost-based options to protect their “private” interests as private companies can. As such, since we have shown that according to the evidence available, the promotion of flexible  and sustainable BPT is, compared to other alternatives, for different particular disease situations, the best solution to promote overall health benefits, then there is a public moral duty to do so. This particular public requirement to promote this specific kind of BPT can perhaps best manifest itself by providing an adapted regulatory framework, so that the full potential of BPT as a locally prepared and tailor-made therapy can finally be realized.In this paper, we have argued that the current state of affairs as described in the introduction is morally unacceptable. We succeeded in underpinning the desirability for developing a flexible and sustainable BPT-adapted regulatory framework with the necessary moral force. The authors are aware that moral arguments in favour of BPT may equally be identified  and apply to other areas of drug development . We argued that the pharmaceutical industry has a moral duty to invest in BPT in view of the social responsibility they need to take. But of equally crucial importance is the role of the competent public authorities to create the appropriate regulatory and legal framework to stimulate companies to invest in BPT. Political representatives and lawmakers have an inevitable, logical responsibility to support health care and welfare. That is what they are for. We identified a shared responsibility making BPT accessible for patients in need. The development and production of BPT products in a pharmaceutical context  requires time. Patients in need have no time. Therefore, lawmakers and regulators need to design appropriate solutions on a short term to buffer for the years needed for companies to develop BPT-based medicinal products. Much more urgent and optimal, regulatory solutions need to be created to allow hospitals to adopt patient-oriented and tailored BPT in a legal way for treating those patients that are waiting to be cured.