Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2′-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.cADPR is a second messenger that releases Ca Pharmaeceptors ,5. Besidiewed in ). In the2+-permeable member of the melastatin subfamily of TRP channels, can be activated by cADPR [50 of 700 µM, while endogenous concentrations are in the low µM range [50 of 70 µM), indicating that cADPR acts upon a binding site distinct from the NUDT9 homology domain (NUDT9H) [In 2005, Kolisek et al. first reported that TRPM2, a nonselective, Caby cADPR . Before,by cADPR . The cADµM range ), but th(NUDT9H) . In the (NUDT9H) ,14 (see (NUDT9H) ).Nematostella vectensis (nvTRPM2) showed that the NUDT9H domain in this channel is not required for activation by ADPR and instead hydrolyses the agonist, which was a first indication that the initial assumption that the NUDT9H domain is solely responsible for gating of TRPM2 is incorrect [Recent developments again raised the question of whether cADPR could be a direct TRPM2 modulator. Seminal work from Kühn and coworkers on the TRPM2 orthologue of ncorrect . The firncorrect . Interesncorrect ). The ADncorrect .These developments led us to revisit the role of cADPR in TRPM2 activation. We tested cADPR, which we checked for purity by HPLC, for potential activation of TRPM2 under a variety of conditions, introduced mutations into the MHR1/2 and NUDT9H domain to analyse their role in agonist activation, and determined the binding of the MHR1/2 domain of TRPM2 to ADPR and cADPR.Since cADPR is prone to both enzymatic and nonenzymatic hydrolysis, we analysed three different lots of cADPR obtained from two different vendors using ion-pair RP-HPLC ,6. These2+ in the pipette solution to 200 nM using EGTA.To test cADPR for its activity towards TRPM2, we used a HEK293 cell line with stable expression of human TRPM2 we generated previously . To avoi50 of 700 µM at room temperature [n = 13). The application of 250 µM ADPR in the pipette, in contrast, resulted in significantly higher outward currents  compared with the buffer control  at 37 °C. Application of 250 µM cADPR did not significantly further increase this current , whereas 250 µM ADPR resulted in a significantly higher current (16.5 nA ± 5.5 nA) compared with the buffer control at 37 °C. Of note, a roughly 18-fold higher current as compared with room temperature control was observed at 250 µM ADPR  but not at 25 °C [ °C but nand Ca2+ ,9,10. Du+ in the bath solution, we replaced the extracellular buffer solution with a buffer solution containing NaCl instead of NMDG. While under these conditions, most of the cells infused with 250 µM ADPR in the pipette solution ruptured during the recording, probably as a result of the rush of inward current, and the infusion of 250 µM cADPR did not result in a significant increase in maximum whole-cell current compared to the buffer control  (To exclude that the lack of effect of cADPR was due to the absence of Na n = 10) c.2+ buffering [2+ buffering in our system and used a nominally Ca2+ free buffer solution with 100 µM EGTA. Under these conditions, the channel behaved somewhat differently; instead of the continuous distribution of current amplitudes we observed in the previous experiments, we noticed a bimodal distribution of current amplitudes when we added nucleotides to the pipette solution. Some cells showed a current slightly higher than the buffer control, whereas other cells exhibited a current that was roughly 100-fold higher. We attributed this behaviour to positive feedback by Ca2+ ions entering through the open channel and sensitizing the channel to ADPR [2+ buffering but leads to an “all or nothing” response when the Ca2+ buffering is low. The threshold for TRPM2 activation by ADPR was apparently between 50 µM and 75 µM, at 50 µM, only 2 out of 10 cells showed a current larger than 1 nA, whereas, at 75 µM, 15 out of 16 cells showed a current of this amplitude. In contrast to what would be expected if cADPR acts synergistically with ADPR [n = 12 vs. w cADPR: 1.1 nA ± 0.9 nA, n = 14) nor at the higher concentration  , whereas the addition of ADPR to the pipette solution significantly increased the current  a. In addto cADPR ,25. Furt current c.In the absence of any observable effect of cADPR on human TRPM2, we were interested in whether 8-Br-cADPR, which had been shown to selectively inhibit activation of the channel by cADPR  and to bWhen 1 mM of either 8-Br-ADPR or 8-Br-cADPR was applied via the patch pipette, they resulted in a slight increase in current compared with the buffer alone e. For 8-Nematostella vectensis (nvTRPM2) [n = 20) or the superagonist 2′-deoxy-ADPR, which resulted in a larger current  (n = 9) (The cryo-EM structure of TRPM2 from zebrafish (drTRPM2) led to the identification of a novel nucleotide-binding site in the N-terminus of the channel  that hadnvTRPM2) . In one nvTRPM2)  (especianvTRPM2) ). To invnvTRPM2)  whereas nvTRPM2) . We conf n = 20) c, as we  (n = 9) c. The po (n = 9) b, indicaE. coli and purified it by affinity chromatography and size exclusion chromatography. The isolated domain was then used to measure the binding of the nucleotides ADPR and cADPR by isothermal titration calorimetry (ITC). While we observed endothermal binding of ADPR to the isolated MHR1/2 domain with a KD of 2.2 µM ± 1.2 µM . InterOur interest in cADPR as a potential TRPM2 agonist was raised by a recent structural study that showed 8-Br-cADPR bound to the N-terminal MHR1/2 domain of TRPM2 . In con2+ buffering, or extracellular cation composition. We also observed no additive/synergistic effect of cADPR when coapplied with ADPR at a subthreshold concentration under conditions of low Ca2+ buffering. These findings are in agreement with the previous reports that hydrolysis of contaminating ADPR using a pyrophosphatase abrogates any effects of cADPR on TRPM2 [m/z 540, the molecular weight of cADPR 541 Da,) also a smaller peak (m/z 558) which the authors attribute to a complex of cADPR + water (541 Da + 18 Da = 559 Da), but which in our opinion more likely is ADPR (m.w. 559 Da). The EC50 value for cADPR in this study was significantly larger than that for ADPR (250 µM for cADPR vs. 40 µM for ADPR), which likely is consistent with ADPR contamination of their cADPR preparation. Our patch clamp experiments under nearly identical buffer conditions  exhibited no activation of human TRPM2 by cADPR.Using the purest cADPR preparation available to us, we tested cADPR for activation of human TRPM2 in whole-cell patch clamp experiments but observed no activation of TRPM2 regardless of temperature, Caon TRPM2 ,14. Yu eon TRPM2 . To copeNematostella vectensis and Danio rerio, there has been some dispute whether this also holds true for the human orthologue of TRPM2. While in their first cryo-EM structure of human TRPM2, Wang et al. did not detect ADPR in the MHR1/2 domain and therefore proposed that the human channel does not require binding of ADPR to this domain [This posed the question of the role of the MHR1/2 domain for the activation of human TRPM2. While the MHR1/2 domain is clearly required for activation of TRPM2 from s domain , Huang es domain , this maIf both domains are required for activation by either agonist, this raises the question about the previous reports on the binding of 8-Br-cADPR to the MHR1/2 domain  and seleIn conclusion, while both nucleotide-binding sites, the N-terminal MHR1/2 domain as well as the C-terminal NUDT9H domain, are required for activation of human TRPM2 by the nucleotide agonists ADPR and 2′-deoxy-ADPR (2dADPR), we found no evidence for a role of the cyclic nucleotide cADPR in the activation of the human channel. The “cADPR antagonist” 8-Br-cADPR inhibits activation of the channel by ADPR, which might reflect hydrolysis to the ADPR antagonist 8-Br-ADPR, and the isolated MHR1/2 domain of TRPM2 binds with high-affinity binding to the TRPM2 agonist ADPR but does not bind to the cyclic nucleotide cADPR.2 and tested for mycoplasma contamination on a regular basis .Wildtype HEK293 cells were cultivated in DMEM  with 10% fetal calf serum , 100 units/mL penicillin and 100 µg/mL streptomycin  For HEK 293 #24 (stably expressing TRPM2) 400 µg/mL G418  were added to the medium. The cells were kept at 37 °C and 5% CO2PO4 and 5 mmol/L tetrabutylammonium dihydrogen phosphate, pH 6.0) with increasing amounts of methanol, resulting in the following gradient: 15% from start to 3 min, 31.25% at 11 min, 50% at 25 min, 50% at 27 min, 15% at 29 min, 15% at 38 min. The flow rate was 0.8 mL/min. Nucleotides were detected by their UV absorption at 260 nm and identified and quantified using external standards. Chromatograms were analysed offline using ChemStation Software .Nucleotides were checked by ion pair reverse-phase HPLC (RP-HPLC) as described before . SamplesConstruction of the expression vector for human TRPM2 (pIRES2-EGFP-hTRPM2) has been described before . The mut5 cells in a cell culture medium as described above without antibiotics. Cells were kept at 37 °C and 5% CO2 overnight.Wild type HEK 293 cells were transfected with either pIRES-EGFP-TRPM2-wt (control), pIRES2-EGFP-hTRPM2-R302A/R358A, or pIRES2-EGFP-hTRPM2-R1404Q and 1 µg of vector DNA was incubated with 5 µL of JetPEI solution  in 150 mM NaCl solution  at a total volume of 250 µL for 30 min. The mix was added to Greiner 35 mm culture dishes  containing 2.5 × 102, 1 mM CaCl2, pH 7.4 at room temperature). For experiments at 37 °C, the temperature dependence of the buffer was taken into account and the pH adjusted to pH 7.63 at room temperature to make sure that the solution had a pH of 7.4 at 37 °C. In some experiments, the NMDG in the bath solution was replaced by 140 mM NaCl. 1.10 mm × 1.50 mm × 80 mm capillaries from borosilicate glass were pulled into pipettes with resistance between 1.5 and 3.5 MΩ using a P-97 horizontal puller  and filled with pipette solution . For the experiments performed at 37 °C, the buffer contained 5.947 mM CaCl2, and the pH was adjusted to 7.42 at room temperature to make sure the solution had 200 nM free Ca2+ and pH 7.2 at 37 °C. Ca2+ concentrations were calculated using MaxChelator. For some experiments, Ca2+ buffering was reduced. In these cases the pipette solution contained 129.3 mM KCl, 10 mM HEPES, 100 µM EGTA, 8 mM NaCl, and 1 mM MgCl2 at pH 7.2.Before the electrophysiological experiments, the culture medium was removed, and cells were washed with the respective bath solution twice. During the experiments, the cells were kept in 1 mL of the bath solution. The standard bath solution was based on NMDG (140 mM N-methyl-D-glucamine (NMDG), 5 mM KCl, 5 mM D-Glucose, 10 mM HEPES, 3.3 mM MgClWhole-cell patch clamp experiments were performed using an EPC-10 amplifier  and PatchMaster software . After the opening of the cells, the holding potential was set to −50 mV, and 90 voltage ramps between −85 and +20 mV (over 140 ms) were applied, one every 5 s. Compensation of series resistance was set to 70%. For the measurements that required temperature control, a peristaltic pump  and a single channel heat controller  were added to the setup, and the cells were continuously perfused with prewarmed bath solution at a flow rate of 0.5 mL/min. The temperature within the dish was checked using a thermocouple thermometer.Adenine nucleotides , 2′-deoxy-ADPR , cADPR , 8-Br-ADPR , 8-Br-cADPR , and cIDPR ) were added to the pipette solution at the indicated concentrations.For each condition, one T25 flask of wild type HEK293 cells was prepared at 75% confluency and transfected using the Lipofectamine LTX and plus reagents  using the TRPM2 expression vectors described in 2+ and Mg2+) and treated with 1 mg/mL EZ-Link Sulfo-NHS-LC-Biotin  for 30 min. Afterwards, cells were removed from the flask using 2 mM EGTA in PBS and collected by centrifugation (500× g). The cells were washed two times with PBS/EGTA, and membrane proteins were extracted using the ProteoExtract Native Membrane Protein Extraction Kit . Total membrane protein samples were collected and stored at −20 °C; membrane extraction samples were mixed with NeutrAvidin Agarose beads  overnight, shaking with 20 rpm at 4 °C, centrifugated  and stored at −20 °C as well.48 h post-transfection, successful transfection was confirmed by checking EGFP fluorescence by fluorescence microscopy. Then cells were washed three times with D-PBS  and separated using a precast 4–15 % SDS gel at 200 V for ca. 40 min until the coloured band left the gel. The gel was transferred to a PVDF-Membrane Immobilon-P via Western blot using 25 mM Tris, 192 mM glycine, 20% methanol, 0.025% SDS transfer buffer. The membrane was cut at the 140 kDa marker band . Both parts were washed three times and blocked overnight using 5% milk powder in TBS-T at 4 °C. The upper membrane part was incubated with rabbit a-TRPM2-antibodies  1:50,000 in TBS-T containing 2.5% milk powder for 1 h at RT. Rabbit α-Na+/K+-ATPase-antibodies  1:10,000 were used on the lower membrane part at the same conditions. Membranes were washed in TBS-T three times for 5 min and incubated with HRP-conjugated goat anti-rabbit antibodies  1:10,000 at the same conditions as before. Membranes were washed three times for 5 min in TBS-T.Both sample groups were mixed with 10 µL SDS sample buffer per sample, heated to 75 °C for 7 min, and centrifugated  was mixed 1:10 with SuperSignal West Pico chemiluminescence substrate , put on the membranes for 5 min and carefully dried. The membrane was photographed at one picture per minute.6-tag. E. coli BL21 Gold (DE3) cells were grown in Terrific Broth medium supplemented with 25 µg/mL kanamycin at 37 °C until a density of 0.8 (measured at 600 nm) was reached. Protein expression was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and carried out for 16 h at 20 °C. The cells were harvested by centrifugation, and lysis was carried out using a high-pressure homogenizer . The His6-tagged protein was purified from the clarified lysate using immobilised metal affinity chromatography (IMAC) and subsequent size exclusion chromatography on a Superdex S200 increase 10/300 column  using HEPES buffer .A pnEK-vH vector was used to express the zebrafish TRPM2 MHR1/2 domain  with an N-terminal HisTM software . Adenine nucleotides were dissolved in HEPES buffer to a concentration of 300 µM and placed in the ITC syringe. Nineteen injections of the ligand (2 µL each) were titrated into 20 µM drMHR1/2 in the ITC sample cell. Each individual injection was interspaced by 150 s, and the stirring speed was set to 750 rpm. The ligand was titrated into HEPES buffer to allow for baseline correction. All measurements were performed as triplicates, and binding curves were fitted with a one-site binding model.Binding assays by isothermal titration calorimetry (ITC) were carried out at 25 °C on a MicroCal ITC-200 isothermal titration calorimeter . Thermodynamic parameters were analysed with the MicroCal ORIGINp values for multiple testing. The data acquired with decreased Ca2+ buffering in the pipette solution showed a bimodal distribution; since both modes showed the skewed distribution, these data were also log-transformed but tested using a nonparametric Kruskal–Wallis test with Dunn’s correction. In the case of the experiments with 8-Br-ADPR/8-Br-cADPR, a threshold was set between the modes, and the distributions were tested pairwise using Fisher’s exact test. In all cases, test results with p < 0.05 were considered to be significant.Statistical analysis of the data was performed using GraphPad Prism . Data in the text are represented as mean ± SEM if not indicated otherwise. Since the currents obtained from whole-cell recordings were significantly skewed towards positive values, they were log-transformed to achieve normality. Normality of transformed data was confirmed by Kolmogorov–Smirnov normality test. Normal log-transformed data were tested by one-way ANOVA, using Bonferroni correction to adjust


However, treatments for wound healing are often difficult and have not yet been fully established. In this study, we investigated the effect of a special paste that can be administered transdermally and holds a non-gaseous carbon dioxide , transforming growth factor (TGF)-β, hypoxia-inducible factor (HIF)-1α, interleukin (IL)-1β, and IL-6 using quantitative real-time polymerase chain reaction, hematoxylin and eosin, and immunohistochemical staining patterns.Methods: Forty-eight Sprague Dawley rats were randomized into control and CO2 group showed accelerated wound healing compared to those in the control group. Furthermore, VEGF and TGF-β were overexpressed, whereas HIF-1α, IL-1β, and IL-6 were downregulated in the rats treated with CO2. Immunohistochemical analysis also revealed similar patterns of expression.Results: Rats in the CO2 paste promoted wound healing by regulating the hypoxic environment, reducing inflammation, and accelerating angiogenesis.Conclusion: Taken together, the CO The skin functions as a protective physical barrier against a variety of environmental insults and is critical for homeostasis . Wound d2) therapy has been developed for use in humans and animal models to evaluate its efficacy in treating various conditions, for example, medical and beauty treatments  × 100.The wounds were photographed 0, 3, 7, 14, and 21 days post-wounding and were measured using ImageJ We measured the mRNA levels of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), transforming growth factor-β (TGF-β), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The rats were sacrificed 3, 7, 14, and 21 days after injury. Tissue samples were collected from around the wound from which total RNA was extracted using TRIzol  and treated using the RNeasy Mini Kit . cDNA was synthesized  using the High-Capacity cDNA (complementary DNA) Reverse Transcription Kit . mRNA levels were quantified using the Step One Real-Time PCR System . Real-time PCR (20 µl) was performed using 0.5 μM forward primer, 0.5 μM reverse primer, 1 µl of template cDNA, and 10 µl (2×) Power SYBR Green Master Mix . The PCR conditions were as follows: 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Target gene expression was normalized to β-actin and fold-change was calculated using the 2-ΔΔCT method . β-actin, VEGF, HIF-1α, and IL-6 primers were obtained from Invitrogen and those for TGF-β and IL-1β were procured from Qiagen. The primer information is presented in Tables Hematoxylin and eosin (H&E) stainingThe tissues were fixed in 4% paraformaldehyde and embedded in paraffin wax. The paraffin-embedded 6-µm thick samples were sectioned using a microtome and stained with H&E. We defined wound healing as a condition indistinguishable from tissues adjacent to the site of the wound. Images of the sections were captured using BZ-X700  at a magnification of ×200.Immunohistochemistry2O2, and incubated overnight at 4℃ with the following primary antibodies in Can Get Signal lmmunostain Solution A : mouse anti-VEGFA antibody  and anti-HIF-1α antibody . The rabbit anti-TGFB1 antibody  was heat-treated with ethylenediaminetetraacetic acid (EDTA) instead of proteinase K. Following washes using phosphate-buffered saline, we incubated the sections with peroxidase-conjugated labeled anti-mouse (cat no. 424131) or anti-rabbit (cat no. 424141) antibodies  for 30 min. Following staining with diaminobenzidine and counterstaining with hematoxylin, the tissue sections were visualized and photographed using the BZ-X700 fluorescence microscope  at a magnification of ×400. The positively stained areas were quantified using the Hybrid cell count BZ-H3C software .Immunohistochemistry was performed using the tissue sections from the wound to detect the protein levels of VEGF, HIF-1α, and TGF-β (10 each). The formalin-fixed and paraffin-embedded skin tissues were pretreated with proteinase K , quenched with 0.05% HStatistical analysisData collection and statistical analyses were performed using Excel-Toukei 2012 . Data have been represented as mean ± standard error. Data from the two groups were analyzed using the Mann-Whitney U-test. A value of p <0.05 was considered statistically significant.Rate of wound healingFigure 2 groups. Three days after the wounds were punched, the rates of wound healing in the control and CO2 groups were 21.04% ± 3.62% and 32.98% ± 4.48%, respectively. On day 7, the rates of wound healing in the control and CO2 groups were 38.02% ± 3.66% and 65.11% ± 2.33%, respectively (p < 0.05). On day 14, the rates of wound healing in the control and CO2 were 78.24% ± 1.75% and 86.57% ± 0.90%, respectively (p < 0.05). On day 21, the rates of wound healing in the control and CO2 groups were 82.74% ± 1.75% and 93.30% ± 0.68%, respectively (p < 0.05). Thus, the rates of wound healing were significantly higher after three days in rats treated with the CO2 paste as compared to those in the control rats. Figure Gene expression profiles2 group as compared to that in the control group after seven days (p < 0.05).VEGF levels peaked seven days after injury Figure . Real-ti2 group than that in the control group . The expression of TGF-β days 3, 7, and 14) was significantly higher in the CO, and 14 H&E staining2 group showed a higher abundance of proliferating fibroblasts. Moreover, fibroblasts were uniformly and densely arranged in the CO2 group as compared to the control group. On day 14, the fibroblast content gradually decreased in both groups. The rats from the control group showed remains of a scar. However, rats in the CO2 group showed better healing around the wound. Rats from the CO2 group showed uniformly arranged collagen fibers after 21 days. These rats only had a scar near the epithelium. In contrast, rats from the control group possessed a lower abundance of collagen fibers and a full scar.Figure Immunohistochemical analysisHIF-1α, VEGF, and TGF-β protein levels in the wounds were assessed using immunohistochemistry. HIF-1α-positive cells were more abundant in the control group across all the time points Figures , 10.2 group showed higher VEGF expression as compared to that in the control group on days 7, 14, and 21 (Figures The CO Figures , 12.2 group showed higher levels of TGF-β as compared to that in the control group across all time points (Figures Moreover, the CO Figures , 14. The2 paste accelerated wound healing by upregulating VEGF and TGF-β and downregulating HIF-1α, IL-1β, and IL-6 in wounded rats.In this study, we have demonstrated that CO2 therapy mechanistically involves the percutaneous absorption of CO2 that enhances vasodilation, blood flow, Bohr effect, and tissue O2 partial pressure. We have previously demonstrated that transcutaneous administration of CO2 results in an artificial Bohr effect by increasing tissue O2 pressure upon CO2 absorption [2 therapy has been used in humans and animal models to study its medical and cosmetic effects [2 system improves blood flow and increases angiogenesis [2 cannot be used in the head and neck region. In this study, we used a CO2 paste for wound healing that may be applied to the head and neck.COsorption . Transcu effects ,13. We hogenesis ,9. GaseoWound healing is a complex process that involves multiple biological processes, such as inflammation, formation of granulation tissue, re-epithelialization, and matrix formation and remodeling . Wound h2 and is involved in transcriptional regulation during hypoxia [2 group failed to exhibit sustained hypoxia.HIF-1α is a protein that senses changes in environmental O hypoxia . Hypoxia hypoxia . Early-s hypoxia . In this2 therapy-induced increase in the expression of VEGF in rat fracture, skin flap, and muscle models [2 group on day 7. This indicates that the CO2 paste stimulates the expression of growth factors. Under hypoxia, VEGF is primarily regulated by HIF-1α [2 [VEGF is produced by endothelial cells, fibroblasts, and macrophages and is important in wound healing. VEGF promotes the early events in angiogenesis, particularly endothelial cell migration and proliferation . VEGF lee models ,9,18. Iny HIF-1α . VEGF isIF-1α [2 . We obse2 group on days 3, 7, and 14.TGF-β is important for angiogenesis and wound healing . TGF-β i2 group on day 7. On day 14, the skin around the wound was replaced with collagen fibers that enabled wound shrinkage. The peak in the expression of TGF-β on day 7 in the CO2 group highlighted accelerated wound healing, implicating the CO2 paste in inducing normal healing. We observed a slight difference between our real-time PCR and immunostaining data for TGF-β. TGF-β mRNA levels peaked on day 3, while its protein levels peaked on day 7. This could be attributed to delayed protein synthesis.Scarless wounds are characterized by a lack of inflammation, regeneration of dermal appendages, and orderly deposition of collagen . Fibrobl2 group than that in the control rats. Therefore, CO2 paste might accelerate wound healing by regulating inflammatory cytokines and hypoxia.An increase in inflammatory cytokines, such as IL-1β, prolongs inflammation and delays healing . Hypoxia2 treatment, such as application time and the number of days of treatment. Finally, the rats of the control group received only wound generation without any control paste. Therefore, the influences of treatment without a CO2 paste were not completely excluded. These limitations should be investigated in further studies.However, this study has some limitations. First, this study involves an animal model and has not been conducted in humans. This is important since rat and human skin are different. Second, this study does not address the optimum conditions of CO2 paste accelerated wound healing by upregulating VEGF and TGF-β and downregulating HIF-1α, IL-1β, and IL-6 in a rat model of skin defect. The application of CO2 paste accelerated normal wound healing by improving hypoxia and angiogenesis and reducing the expression of inflammatory cytokines. Further experiments, including human clinical studies, are needed to investigate the efficacy of CO2 paste on skin wounds. This will enable the development of novel strategies for patients with skin defects in the future.In summary, this study demonstrated that CO


P < 0.05); the expression of S1PR2 increased 12 h after MCAO modeling and returned to the normal level on the 5th day, and the content of MMP9 protein in brain tissue of the test group was significantly lower than that of the control group (P < 0.05); the mRNA levels and SODs activity of SOD1, SOD2, and SOD3 in the test group were higher than those in the control group (P < 0.05). Therefore, miR-149-5p played a neuroprotective role by regulating S1PR2 to change the expression levels of SODS and MMP9.To investigate the effect of miR-149-5p on sphingosine-1-phosphate receptor 2 (S1PR2) expression level and contents of matrix metalloproteinase (MMP-9) and superoxide dismutase (SOD) in the pericytes after acute cerebral ischemia reperfusion in rats, so as to clarify the neuroprotective molecular mechanism induced by miR-149-5p and provide references for the treatment of neurological diseases, 60 male SD rats aged 7–8 weeks were selected and divided randomly into test group  model) and control group (no modeling). Rat pericytes and peripheral cerebral infarction tissues were collected 12 h, 1 d, 3 d, 5 d, and 7 d after MCAO modeling, respectively. The pericytes were identified by immunofluorescence assay (IFA) and transfected with miR-149-5p. Fluorescence quantitative PCR (FQPCR) and Western blot were adopted to detect S1PR2 expression level. The expression of S1PR2 in MCAO model rats was detected by IFA. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were used to detect the changes of MMP9 protein and mRNA levels of SOD1, SOD2, and SOD3 in brain tissue. The results showed that mRNA level and protein expression level of S1PR2 in the test group were higher than those in the control group three days after MCAO modeling (  IschemiThe endothelial part of BBB is the main site of reactive oxygen species (ROS) production. Current studies found that inflammatory mediators, vascular endothelial growth factors, and micro-RNAs (miRNAs) all had certain effects on BBB . StudiesBased on the above speculation, the rat model of acute ischemia reperfusion was established to investigate the effects of miR-149-5p on the expression levels of S1PR2 and SODS at the cellular and molecular levels. This work aimed to analyze the regulatory effect of miR-149-5p on pericytes S1PR2 after acute ischemia reperfusion in rats and whether it was involved in the neuroprotective mechanism, so as to provide reference basis for the treatment of late nervous system diseases.2.2.1Sixty healthy male SD rats aged 7–8 weeks, weighing 265–305 g were selected, with 5 in each cage in SPF laboratory. The room temperature was 25°C, and relative humidity was about 55%. They were given free drinking water and diet under 12 h of light. After 2 weeks of adaptive feeding, all rats were randomly divided into two groups: test group (30 rats) and control group (30 rats). I/R model was established in the test group and not in the control group. All animal experiments in this study were approved by the experimental animal control committee, and the experimental methods were conducted in accordance with the approval guidelines.2.2The middle cerebral artery occlusion (MCAO) model in rats was established by reference to the improved line occlusion method and modified based on that . Rats we2.32), the liquid was changed once for 3 days. The cultured pericytes were identified by IFA. The test group pericytes Lipofectamine 2000 were transfected with miR-149-5p at 50 nmol/L . Transfection was performed according to the instructions of the transfection kit. The cells were placed in 96-well plate one day before transfection, and the cell density was between 40% and 60%. After the cells were adherent to the wall, serum-free medium was added to culture them overnight at 37°C, and the cell density at transfection was 60–70%.Three rats were sacrificed 12 h, 1 d, 3 d, 5 d, and 7 d after the establishment of the I/R model, and pericytes and brain tissue of all rats were collected. Rat pericytes were obtained by referring to the previous method  and modi2.4After the rats were killed, the brain tissues were stripped off and washed with normal saline precooled at low temperature, and were put into a homogenizer and cut into pieces as far as possible. The total RNA of pericytes or brain tissue was extracted by TRIzol method, and the extracted total RNA was detected by agar-gel electrophoresis. Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR retroviruses kit (Takara) was adopted for reverse transcription. The reverse transcription was performed at 37°C for 15 min and 85°C for 5 s. Real-time FQPCR primer for synthesis of S1PR2 gene was S1-F: 5ʹ-AAATCCAATACGGTACAAACG-3ʹ; S1-R: 5ʹ-GGTCA GACAGCA CCCACA-3ʹ. Real-time FQPCR primers for synthesis of SOD1 gene was SO1-F: 5ʹ-GAGACCTG GGCAATGTGACT-3ʹ; SO1-R: 5ʹ-GTTTACTGCGCAATCCCAAT-3ʹ. Primer for synthesis of SOD2 was SO2-F: 5ʹ – CCGAGGAGAAGTACCACGAG-3ʹ; SO2-R: 5ʹ- GCTTGATAGCC TCCAGCAAC-3ʹ. Primer for synthesis of SOD3 was SO3-F: 5ʹ- ATCCCACAAGCCCCTAGTCT-3ʹ; SO3-R: 5ʹ- GTGCTATGGGGACAGGAAGA-3ʹ. GAPDH was taken as internal reference, primer of GAPDH gene was Fm: AGTTCAACGGCACAGTCAAGG; Rm: CAGCCTTCTCCATGGTGGTG. Each sample was amplified with the above three pairs of primers, and each reaction was repeated for three times.2O was added to make up the total volume to 20 μL. The amplification conditions of the PCR reaction were as follows: 94°C for 5 min; 94°C for 10 s, 60°C for 20 s; 72°C for 30 s; with a total of 40 cycles; then 72°C for 2.5 min, and 40°C for 2.5 min. The reaction was carried out in Exicycler TM 96 fluorescent quantitative PCR instrument. The 2−ΔΔct method was used to calculate the relative expression level [The qRT-PCR reaction system is as follows: 1 μL cDNA, 0.3 μL SYBR Green, 10 μL 2× Taq PCR Master Mix, 0.5 μL forward primer, and 0.5 μL reverse primer. Then, ddHon level .2.5The peripheral infarcted tissue after acute ischemia reperfusion was put into a homogenizer and cut into pieces as much as possible. 1 mL of pre-configured lysate (RIPA protein lysate: protease inhibitor PMSF = 1:100) was added for grinding, so that the tissue was fully lysed to extract the total protein. The total protein was determined by bicinchoninic acid (BCA) assay. 12% Tris-glycine gradient gel was used to separate cell proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V−120 V. The protein was transferred to the polyvinylidene fluoride (PVDF) membrane by the wet transfer method at 4°C and 80 V. Then, it was sealed with 5% skimmed milk at room temperature for 1 h. Protein was transferred to polyvinylidene fluoride (PVDF) membrane by wet transfer method, blocked by 5% skimmed milk, and incubated with antibodies of S1PR2 and GAPDH diluted by 1:1000 at 4°C overnight. Protein was washed thoroughly with tris-buffered saline tween (TBST) for three times, 10 min for each time, and incubated with horseradish peroxidase-coupled secondary antibody at room temperature for 2 h; TBST was used to wash it thoroughly for three times, 10 min for each time, and color developing solution was added. The image of protein was exposed automatically on the developer, and the grayscale scan was conducted. The protein expression was analyzed according to the gray value.2.6Immunohistochemical assay was used to detect the protein content of MMP9 in the brain tissue of the test group and the control group, and anti-MMP9 antibody was taken as the primary antibody. Immunohistochemical operations were carried out in accordance with routine operations . The exp2.7Rats were sacrificed 12 h, 1 d, 3 d, 5 d, and 7 d after I/R model was established, respectively. Perfusion was performed with precooled PBS and precooled 4% paraformaldehyde in turn. Brain tissue and pericytes were collected. The brain tissue was dehydrated and sectioned (20 μm), 5% bovine serum (with 0.3% Triton X-100) was added for sealing for 1 h after washing with PBS for three times, and incubated with primary antibody overnight at low temperature. Washed with PBS for three times, the tissue was added with fluorescently labeled secondary antibody and incubated at room temperature for 2 h. 4ʹ, 6-diamidino-2-phenylindole (DAPI) staining was performed for 10 min, and the sections were sealed. Photographs of the tissue were taken with the laser scanning confocal microscope.2.8After the rats were killed, their brain tissue was taken out and the pre-cooled 50 mmol/L Tris-HCl (pH = 7.0) was added into the homogenizer for tissue homogenization. After centrifugation, supernatant was taken and SODs enzyme activity in brain tissue was detected by the SODs kit . Specific operation steps were carried out in accordance with the instructions.2.9t-test was used for measurement data subjected to normal distribution, Wilcoxon test was used for measurement data subject to normal distribution, and P < 0.05 was considered statistically significant.SPSS 19.0 was adopted for data statistics and analysis. Measurement data were expressed as mean plus or minus standard deviation , and SODs in pericytes after acute cerebral ischemia and reperfusion in rats, so as to clarify the neuroprotective molecular mechanism induced by miR-149-5p. In this study, a middle cerebral artery occlusion (MCAO) model was established in healthy male SD rats. Rat pericytes were collected and transfected with miR-149-5. Then, fluorescence quantitative PCR was used to analyze the changes of S1PR2, SOD1, SOD2, and SOD3 mRNA levels. Western blot and immunofluorescence were used to detect the protein expression of S1PR2. In addition, immunohistochemistry was used to detect the changes of MMP9 protein levels in brain tissues, and the activity of SODS enzyme was detected .Figure 13.2The isolated rat brain microvascular pericytes (BMPs) were cultured in vitro. The pericytes were found to be radially or elliptically arranged during culture. With the increase of cell-culture algebra, positive platelet-derived growth factor receptor-β (PDGFR-β) increased, other cells such as neurons and microglia cells were fewer and fewer. After three times of subculture, almost no other cells were found, indicating that pericytes could be purified by subculture. The identification results of the third generation after subculture by α-smooth muscle actin (α-SMA) and PDGFR-β were shown in 3.3P < 0.05). The mRNA level of S1PR2 reached the highest value (8.01 ± 0.98) on the first day after MCAO modeling, showed a downward trend on the third day, and decreased to the lowest level on the fifth and seventh days, with no significant difference compared with the control group (P > 0.05). The S1PR2 protein expression level of rats in the control group remained unchanged during the test, while that of the test group was significantly higher than that of the control group at 12 h after MCAO modeling (P < 0.05). On the first day after MCAO modeling, the expression of S1PR2 protein reached the highest value (4.13 ± 0.27), which was significantly different from that of the control group (P < 0.05). The expression of S1PR2 protein decreased to the lowest level on the seventh day after MCAO modeling, with no significant difference compared with the control group (P > 0.05).The expression level of S1PR2 in pericytes of rats after acute I/R was detected by quantitative reverse-transcription PCR (qRT-PCR) and Western blot, respectively. The results were shown in modeling , with a 3.4Immunofluorescence staining was adopted to detect the expression of S1PR2 in peripheral infarcted tissues of MCAO rats. And PDGFR-β was taken as the pericytes marker, and the results were shown in 3.5P < 0.05).IHC was adopted to stain the MMP9 protein in the brain tissues of the two groups of rats, and the results were shown in 3.6P < 0.05).The mRNA levels of SOD1, SOD2, and SOD3 in the brain tissues of rats in the test group and the control group were detected, and the results are shown in 3.7P < 0.05).The SODs enzyme activity in the brain tissue of the test group and the control group was detected and compared. The results were shown in 4.P > 0.05), which was consistent with the research of Matsumoto et al. (2020) [P < 0.05), which was consistent with the current results, indicating that miR-149-5p had a certain effect on the expression of MMP9 protein.After acute ischemic reperfusion, BBB would be damaged in certain degrees, leading to intracranial hemorrhage, cerebral edema, and other complications . A large. (2020) , who dee. (2020) . The res. (2020)  found th. (2020) . The res2- accumulation in cerebral vessels. Mice lacking the SOD1 gene had elevated O2- levels and impaired cerebrovascular diastolic function. Excessive destruction of O2- by SODs can increase the generation of dismutase products and powerful cerebrovascular diastolic H2O2 [P < 0.05); the activity of SODs enzyme in test group was significantly higher than that in control group (P < 0.05), which were consistent with the current studies that increased SODs expression in brain tissue could significantly reduce nerve injury. The results of this study indicated that enhanced SODs activity could reduce ROS levels in rats after ischemia reperfusion and played a protective role on nerves.The change of SOD expression in the brain had different degrees of influence on the local ROS level in brain. Studies showed that after activation, SODs played a protective role on cerebral nerves, enhanced SODs activity, and caused a significant decrease in ROS content . Some stlic H2O2 . The res5.In this study, a rat model of acute cerebral ischemia reperfusion was established, and miR-149-5p was infected with pericytes in the test model group, so as to detect the regulatory and neuroprotective effects of miR-149-5p on the expression level of S1PR2 in pericytes. The results showed that miR-149-5p significantly increased the expression of S1PR2, SOD1, SOD2, and SOD3, and significantly decreased the expression of MMP9 in pericytes after ischemia reperfusion, which alleviated BBB permeability and played a protective role on nerves. However, there were still some deficiencies in this study that only the regulation of miR-149-5p on S1PR2 in pericytes had been studied, while the specific pathways of the two had not been discovered and it was not clear whether miR-149-5p acted directly on S1PR2. In the later study, the regulation pathways of miR-149-5p on S1PR2 would be analyzed. In conclusion, miR-149-5p had a regulatory effect on S1PR2 of pericytes after acute cerebral ischemia reperfusion in rats and had a neuroprotective effect by regulating the expression levels of MMP9 and SODs.


However, the clinical application of second-generation TSPO ligands as biomarkers is limited because of the presence of human rs6971 polymorphism that affects their binding. Here, we describe the ability of a new TSPO ligand, [3H]PK 11195 using membrane proteins isolated from 293FT cells expressing TSPO-wild type (WT) or TSPO-mutant A147T (Mut), corresponding to a high-affinity binder (HAB) and low-affinity binder (LAB), respectively. Molecular docking was performed to investigate the interaction of BS224 with the binding sites of rat TSPO-WT and TSPO-Mut. We synthesized a new 18F-labeled imidazopyridine acetamide ([18F]BS224) using boronic acid pinacol ester 6 or iodotoluene tosylate precursor 7, respectively, via aromatic 18F-fluorination. Dynamic PET scanning was performed up to 90 min after the injection of [18F]BS224 to healthy mice, and PET imaging data were obtained to estimate its absorbed doses in organs. To evaluate in vivo TSPO-specific uptake of [18F]BS224, lipopolysaccharide (LPS)-induced inflammatory and ischemic stroke rat models were used.An in vitro competitive inhibition assay of BS224 was conducted with BS224 regardless of polymorphisms. With optimal radiochemical yield  and purity (> 99%), [18F]BS224 provided a clear visible image of the inflammatory lesion with a high signal-to-background ratio in both animal models  without skull uptake.BS224 exhibited a high affinity (18F]BS224 may be a promising TSPO ligand to gauge neuroinflammatory disease-related areas in a broad range of patients irrespective of the common rs6971 polymorphism.Our results suggest that [The online version contains supplementary material available at 10.1007/s00259-021-05617-4. The translocator protein 18-kDa (TSPO) is expressed predominantly in steroidogenic tissues . In vivo18F) or carbon-11 (11C) into different structural classes of chemical compounds, such as isoquinoline carboxamides  [d2) [11C]PK 11195 is not dependent on the rs6971 polymorphism [11C [18F-labeled form [18F]CB251 as a new TSPO ligand [18F]CB251 has a great potential in assessing neuroinflammation as it displays higher TSPO affinity and selectivity, regardless of the rs6971 polymorphism phenotype, compared with previously described TSPO ligands [18F]CB251 PET showed an unspecific uptake in the skull in our preclinical studies due to defluorination of the parent molecule. This skull image will hinder the exact quantification of TSPO expression in the clinical brain images, because signal can also be detected in the adjacent brain regions such as the cortex, due to a spillover effect pyridine analog, named [18F]BS224, as it was expected to have the same biological properties as [18F]CB251 due to the molecular similarity between fluorine and fluoroethanol  , aryloxy , arylox95) BS224, was successfully synthesized using nucleophilic aromatic substitution (SNAr) 18F-labeling method with boronic acid pinacol ester 6 or iodotoluene tosylate precursor 7.The detailed experimental procedures for the synthesis of the authentic compound  in 18O-enriched water was diluted with deionized water (900–950 µL) and passed through a Chromafix-HCO3 cartridge (pre-activated with 2 mL of ethanol and 5 mL of water). The captured 18F ion on the cartridge was eluted with a mixture solution of 18-Crown-6 (0.18 mg for 6 or 3.5 mg for 7) dissolved in 1 mL of methanol, and CsHCO3 (0.10 mg for 6 or 1.1 mg for 7) in 10 µL of water. The eluted mixture with 18F was dried by azeotropic distillation with acetonitrile (CH3CN) twice (0.3 mL × 2) at 90 °C under a stream of nitrogen gas. A solvent mixture with 6 , Cu(OTf)2 , and pyridine  was added to the reaction vial containing the dried 18-Crown-6/18F−Cs+ complex (18.5–1110 MBq), followed by heating at 110 °C for 10 min.Radiochemistry of BS224 (TR = 28 min) collected from the HPLC system was diluted with water (20 mL). The HPLC solution was exchanged with 10% ethanol-saline solution using a C18 plus Sep-Pak cartridge for the preclinical studies.For precursor 18F]BS224 was measured five times by mixing a solution of [18F]BS224 (7.4 MBq) in 5% ethanol-saline (10 μL) with phosphate-buffered saline  and n-octanol (5 mL) in a test tube. After vortexing for 1 min, each tube was placed on a table at 25 °C for 3 min. Samples of each phase (100 μL) were counted for radioactivity using a gamma-counter. Log D is expressed as the logarithm of the ratio of the count of n-octanol to that of PBS.The lipophilicity (log D) of [18F]BS224 in 5% ethanol-saline  was incubated with human serum (1.8 mL) from a healthy volunteer for 120 min at 37 °C. Aliquots (0.2 mL) were collected at 0, 15, 30, 60, and 120 min and mixed with acetonitrile (0.2 mL), and then centrifuged at 3500 rpm for 5 min. The resulting supernatants were analyzed using a radio-TLC scanner with the developing solvent (50% ethyl acetate-hexane).To assess in vitro serum stability, [fp) of [18F]BS224 was estimated using an ultrafiltration method [18F]BS224 (0.37 MBq) in 5% ethanol/saline (10 µL) was added to each plasma samples  and saline , and then the samples were incubated at 37 °C for 10 min. The mixtures were transferred to the Amicon® Ultra 30 K device , and centrifuged at 12,000 rpm for 15 min. The radioactivity of [18F]BS224 before  and after filtration  was measured using a gamma-counter. The plasma-free fraction (fP) was calculated as fp = /.Plasma protein binding (PPB) was measured using two plasma samples obtained from five rats and a healthy volunteer. Plasma-free fraction (n method . [18F]BS18F]BS224 (37 MBq) was intravenously injected into the rat tail vein. The rats were sacrificed by carbon dioxide inhalation at 30 and 90 min (n = 3 for each time point) after [18F]BS224 injection. The tissues  were collected and homogenized with 1:1.4 v/v PBS (pH 7.4) and CH3CN (2 mL), and then centrifuged at 3000 rpm for 15 min at 4 °C. Blood (3 mL) was collected into a Li-heparin tube and then centrifuged at 3000 rpm for 15 min at 4 °C. After centrifugation, the supernatant (1 mL) was collected and mixed with CH3CN (1.4 mL). After centrifuging again, the supernatant was analyzed using a semi-preparative HPLC system . The percentages of [18F]BS224 (TR = 14.5 min) to the total radioactivity (corrected for decay) on the HPLC chromatogram was calculated as % =  × 100.In vivo metabolic stability test was performed using male Sprague–Dawley rats . [3H]PK 11195 and [3H]flunitrazepam binding from rat cerebral cortex samples; the result is expressed as inhibition constant (Ki). The binding assay was performed following a previously published procedure PK 11195 (0.019 MBq/100 μL) in membrane proteins (100 μg/100 μL) that were isolated from 293FT cells expressing TSPO-WT or TSPO-Mut, which represented the HAB or LAB phenotypes, respectively.The effect of the rs6971 polymorphism on the binding affinity of BS224 was evaluated using an in vitro competitive inhibition assay  were used to perform in vivo organ distribution and dosimetry studies . Male Sprague–Dawley rats  were used to create rat models of neuroinflammation and ischemic stroke (n = 4 for each model). An acute neuroinflammation rat model was generated by injecting lipopolysaccharides  into the right striatum of rat brain according to our previous protocol BS224 (30.2 ± 1.6 MBq), followed by a CT scan. All data were reconstructed into three-dimensional images using the iterative 3D ordered subset expectation maximization (OSEM) algorithm to generate dynamic PET images.The PET imaging study was performed using a dedicated animal PET/CT scanner  with a 10-cm axial field-of-view (FOV) and a 12-cm transaxial FOV. Before PET imaging, the rats were anesthetized with 2% isoflurane. LPS-induced neuroinflammatory and ischemic stroke rat models (ND) was also calculated as a quantitative value, which represented the receptor binding with the radioligand. The BPND was estimated using a simplified reference-tissue model (SRTM) by taking the TAC of the contralateral side as the reference region BS224 (Table 3), the 18F-labeling of 6 was carried out via copper-mediated 18F-fluorination. The use of tetrakis(pyridine)copper(II) triflate catalyst for aromatic 18F-fluorination failed to yield [18F]BS224, but its individual agents  triflate in the presence of pyridine) produced the desired product with 9.3% yield (entries 1 and 2). When a considerably lower amount of the 18-Crown-6 (0.7 μmol)/CsHCO3 (0.5 μmol) complex was used, the yield of [18F]BS224 increased linearly (up to 65%) depending on the solvent (entries 3–5). In contrast, higher amounts of Cu(OTf)2 and pyridine (Entry 6) did not increase the radiochemical yield (RCY) of [18F]BS224 compared with their optimal amounts (3.3 μmol of Cu(OTf)2 and 0.1 mmol of pyridine). The synthesis of [18F]BS224 using 7 in N,N-dimethylformaide (DMF) or CH3CN solvent at 100 ℃ resulted in a poor RCY of the final product (entries 7 and 8). These low yields of [18F]BS224 were easily overcome when the reaction temperature was increased up to 140 ℃ with CH3CN in a pressure tube (entry 10). The radioactivity of 18F for the optimization of the reaction condition showed in Table 18F]BS224 using 6 included pre-purification on a Sep-Pak cartridge and purification via HPLC and resulted in 39 ± 6.8%  RCY and with high radiochemical purity  ranged from 315 to 472 GBq/µmol. The identity of [18F]BS224 was confirmed by co-injection with the authentic compound 1 using analytical HPLC  compared with in the plasma  of [18F]BS224 was 6.4 ± 0.3 and 4.9 ± 0.5% in rat and human, respectively  of BS224 was highly accumulated in the peripheral organs such as the heart, lung, kidneys, and liver in healthy mice at early time. Low Cmax and AUC values of the bladder, compared with those of the kidneys, suggested that [18F]BS224 was not excreted into the bladder via the urine  and contralateral  regions early during imaging. During the late phases of imaging, especially for accumulated [18F]BS224, its rapid wash-out was observed in the contralateral region. Finally as shown Fig. 418F]BS224 in the ipsilateral region to that in the contralateral region was 2.646 ± 0.336 (P = 0.0318) at 90 min. The AUC value of the ipsilateral region was 2.15-folds higher (P = 0.0208) (AUC = 68.03 ± 9.42) than that of the contralateral region (AUC = 31.68 ± 2.76). BPND of [18F]BS224 was 1.43 ± 0.17. Next, PET studies of [18F]BS224 were also performed in the ischemic stroke rat model . Similar to the results in the LPS-induced neuroinflammatory rat model, a high accumulation of [18F]BS224 was observed in the ipsilateral  and the contralateral region  at early during imaging, followed by rapid wash-out in the contralateral region  at 90 min  than that of the contralateral region (AUC = 18.84 ± 2.79) in the ischemic stroke rat model. The results obtained from the PET images of [18F]BS224 were consistent with the TTC staining result -[18F]GE387, (R)-[18F]NEBIFQUINIDE, [18F]LW223, and [18F]PBR316). One of these is an acetamidobenzoazolone analog; [18F]FEBMP was evaluated for PET imaging of neuroinflammation in rat and in vitro autoradiography on postmortem human brains. In the in vitro autoradiographic assay of human brain tissues, [18F]FEBMP showed a LAB/HAB ratio of 0.9. TSPO rs6971 polymorphism appeared to have a little influence on the binding sites for [18F]FEBMP, but this radioligand showed a metabolic stability not eligible in rats as its radiolabeled metabolite rapidly increased in the plasma and its was also found in the brain tissue GE387 overcame the binding sensitivity of the tricyclic ligand [18F]GE180; however, further investigation of each enantiomer for brain imaging remained [R)-[18F]NEBIFQUINIDE and [18F]LW223, two examples of 18F-labeled analog of PK11195, presented insensitivity to the rs6971 polymorphism, but PET imaging of the neuroinflammatory model has not been performed [18F]PBR316 showed a nanomolar affinity for TSPO, and low sensitivity to rs6971 polymorphism with a LAB/HAB ratio of 1.5. The metabolite analysis showed that the principal radioactivity was in the brain , owing to the original radiotracer pyridine analog [18F]BS224 as a new-generation TSPO PET ligand for neuroinflammation imaging.Recently, several n tissue . -[remained . Other lerformed . [18F]PBiotracer . In this18F-fluorination on the right phenyl of BS224, the electrophilic 18F-labeling method is not suitable for a TSPO PET ligand because of a low molar activity (AM) of the final product. Furthermore, this imidazopyridine substituent at the para position would not provide sufficient activation for the incorporation of 18F using conventional SNAr 18F-labeling methods [NAr 18F-labeling precursors such as hypervalent iodines [NAr 18F-labeling strategy to overcome the radio-synthetic problem of [18F]BS224. Based on well-established aromatic 18F-labeling methods for fluoroarenes including those described in our previous studies [6 and 7) and compared their potency on aromatic 18F-fluorination of [18F]BS224, as shown in Table 6, individual addition with Cu(II) triflate and excess pyridine was more efficient for the aromatic 18F-fluorination of [18F]BS224 than Cu(Py)4(OTf)2. In addition, the highest 18F-fluorination efficiency was obtained when DMF and a lower amount of 18-Crown-6/CsHCO3 were used. These increased 18F-fluorination effects might be due to the additive pyridine that has a stabilizing effect on the complex of metal 18F in the presence of minimum amounts of 18-Crown-6/CsHCO3 BS224 and produced approximately 2.5-fold higher RCY than that obtained under the final optimized conditions for precursor 7. Indeed, even though a reasonable RCY was achieved using 7 as the precursor, precursor 6 was considerably superior in terms of yield and AM of [18F]BS224. The partition coefficient (log D = 2.78 ± 0.04) value of [18F]BS224 was close to that of [11C]PBR28 (log D = 2.82 ± 0.07) among the aryloxyanilide derivatives F-DPA was shown much higher metabolic stability than aliphatic 18F-labeled form, [18F]DPA-714 which suffers O-deethylation in vivo environment in the rat brain. In our study, aromatic 18F-labeled form, [18F]BS224 also showed high in vitro serum stability and in vivo metabolic stability in rat organs, except in the liver and plasma at 90 min. In particular, we measured the radioactivity in the femur by gamma counting to estimate in vivo defluorination of [18F]BS224 in this experiment, but, unlike that in other organs, no radioactivity could be detected in the femurs. Our results suggested that [18F]BS224 has high chemical stability due to fluorine-18 attached to the aryl ring to combat defluorination and no polar radiometabolites are expected to cross the blood–brain barrier. Both values (fp = 6.4 ± 0.3% in rat and 4.9 ± 0.5% in human) indicate that [18F]BS224 has a relatively lower %PPB value than that reported for [11C]PK 11195 (fp = 1% in human) [fp values of [18F]BS224 were comparable with the reported fp values of other second-generation TSPO ligands  BS224 and explain its low sensitivity to the rs6971 polymorphism. The main advantage of this approaches is that it is easier to secure the polymorphic target proteins (A147A and A147T) when compared with human blood and tissues samples generally used to isolate polymorphic TSPOs. Here, we developed a cell system using human embryonic kidney cell lines (293FT) which ectopically express TSPO A147A and A147T leading to higher yield of polymorphic proteins. These cells also allow us to overcome the limitation of having to recruit human volunteers who express the rs6971 polymorphism. Indeed, only 4% of Japanese and 2% of Han Chinese are reported to express the rs6971 polymorphism in the now retired HapMap Resource (http://hapmap.ncbi.nlm.nih.gov), so we could assume that it would be extremely difficult to recruit LAB donors within less than 3% of Korean population to test the in vitro sensitivity of BS224 to the rs6971 polymorphism.The in vitro experiments performed with membrane proteins isolated from 293FT cells and molecular docking simulations allowed us to evaluate the binding capacity of the radiotracer BS224 between LAB and HAB showed the favorable insensitivity to the rs6971 polymorphism. Although our system may overcome the difficulties of in vitro screening using human samples, evaluation of [18F]BS224 in humans should be performed in subjects with polymorphic TSPO phenotype as a further study.As results, the obtained ICn tissue , 31, 46.18F]BS224 for TSPO is not affected by the rs6971 polymorphism, in agreement with the molecular docking simulations. The performed docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low sensitivity of BS224 to the rs6971 polymorphism. Importantly, an in-depth visual inspection of the developed homology models clearly showed that, upon mutation, N151 is subjected to a conformational rearrangement that place its side chain within the binding site  [18F]BS224 is expected to be lower than that of [18F]DPA-714 (17.2 µSv/MBq) in healthy human [11C]PBR-28 (6.6 µSv/MBq) and [11C]DPA-713 (5.9 µSv/MBq) BS224 TSPO PET imaging was conducted at 4 day post-LPS injection and 11 day post-MCAO surgery. Preclinical in vivo images of [18F]BS224 in rat models demonstrated that [18F]BS224 provided a clear visualization of inflammatory lesions compared with that in the contralateral sides. Based on reported results, [18F]BS224 shows relatively high levels of BPND compare with [11C]PK 11195 in both models [ND value (1.43 ± 0.17) of [18F]BS224 in LPS-induced neuroinflammatory rat models is comparable to those of other second-generation TSPO ligands such as [11C]PBR28 and [18F]DPA-714 [ND of the aliphatic 18F-labeled imidazolpyridine analogue [18F]CB251 was slightly higher than that of the aromatic 18F-labeled imidazolpyridine analogue [18F]BS224 in the same model which was produced in the same laboratory. The BPND value of [18F]CB251 was 1.83 ± 0.18, which is comparable with that of [11C]PBR28 (BPND 1.55 ± 0.41) in PET imaging study using the same animals. However, [18F]CB251 PET exhibited an unspecific uptake in the skull due to defluorination in vivo environment. For this reason, we designed and prepared the aromatic 18F-labeled form, [18F]BS224 in this study. In case of [18F]GE-180, the BPND seems to be relatively high in the LPS-induced inflammatory and ischemic stroke rat models [18F]GE-180 was suffering from issues. One is the binding sensitivity to the rs6971 polymorphism (Ki ratio of LAB/HAB = 5.1) and another is low BBB penetration BS224 in two different inflammatory animal models using the SRTM approach to provide a comparison the BPND of [18F]BS224 with our previous results. However, TSPO is constitutively expressed in the brain and therefore the use of the contralateral side as reference region for the ipsilateral side might not be ideal to quantify TSPO PET image in the brain. Therefore, the use of classical compartmental modeling or endothelial modeling of TSPO PET imaging [18F]BS224 PET in a more generalized setting before clinical study. Moreover, it will be of more interest to evaluate a subsequent comparative study of [18F]BS224 with reported TSPO ligands in preclinical or clinical study to prove beyond a reasonable doubt to be a next-generation TSPO ligand.Over the years, various quantification approaches and methods for parameter estimation have been used for the kinetic modeling of TSPO PET in preclinical and clinical studies , 62. Ful imaging , 65 need18F]BS224 could display high signal-to-background ratio in brain imaging because of the low effect of blood pools during early imaging and the absence of skull uptake due to the high in vivo stability of aromatic fluorine. Taken as a whole, these data can represent a valuable starting point for designing the next generation of TSPO ligands.The results in LPS-induced inflammation and ischemic stroke rat models suggested that [18F]BS224, using two different precursors. In aromatic 18F-fluorination of [18F]BS224, boronic acid pinacol ester, precursor 6, showed higher RCY and required relatively milder reaction conditions than iodotoluene tosylate, precursor 7. Results of docking simulations offered a molecular rationale for the low sensitivity to the rs6971 polymorphism exhibited by this new TSPO PET ligand. The LAB/HAB IC50 ratio indicated that the binding affinity of BS224 is less affected by LAB and is comparable to that of the well-known PK 11195. Our PET imaging results suggest that [18F]BS224 displays a high target to background ratio during early imaging of lesioned brain regions with abnormal TSPO expression and provides a clearly visible image of the inflammatory lesions in both animal models without skull uptake. Based on these findings, we do believe that [18F]BS224 might be a promising next-generation TSPO PET ligand to gauge neuroinflammatory disease-relevant areas in a broad range of patients irrespective of the common rs6971 polymorphism.We successfully synthesized a new TSPO PET ligand, [Supplementary file1 (DOCX 4279 kb)Below is the link to the electronic supplementary material.


Novel inhibitor discovery against key factor in WNT pathway is important for CRC treatment and prevention.  Colorectal cancer (CRC) is among the top three gastrointestinal malignancy in morbidity and mortality. The abnormal activation of Wnt/β knockdown.  Cell proliferation was detected after hydroxyphenyl butanone treatment in human colorectal cancer HCT116, LOVO, and normal colonic epithelial NCM460 cells. Colony formation, cell invasion ability, and cell cycle were detected with and without GSK-3β in Wnt/β-catenin pathway and inhibits malignant biological manifestations of cell proliferation, colony formation, and invasion. The inhibition in the high concentration group is stronger than that in the low concentration group, and the antitumor effect is different for different tumor cells. Under the same concentration of natural hydroxyphenyl butanone, the inhibition on normal colonic epithelial cells is significantly lower than that on tumor cells. The natural hydroxyphenyl butanone with medium and low concentration could promote the proliferation of normal colonic epithelial cells.  Hydroxyphenyl butanone induces cycle arresting on G1-S phase of colorectal cancer cell line through GSK3β and revealed the mechanisms underlying the inhibitory effects in colorectal cancer. This study illustrated natural hydroxyphenyl butanone as new inhibitor of GSK3 The abnormal activation of Wnt/β-catenin pathway is considered to be a key factor in the occurrence and development of CRC [β-catenin pathway [Colorectal cancer (CRC) is one of the most common gastrointestinal malignancy. The morbidity and mortality of CRC are among the top three malignant tumors –3. Althot of CRC . In addiβ-catenin is one of the most critical factors to activate Wnt pathway. After targeted for phosphorylation of the N-terminal by glycogen synthase kinase 3β (GSK-3β) at Ser33/Ser37/Tr41 residues, the cytoplasmic level of β-catenin is regulated by ubiquitin-mediated proteolysis. The phosphorylation of β-catenin by GSK-3β occurs in an Axin-APC complex [β activity presents a negative correlation with the presence of Wnt pathway [APC is the key regulator of Wnt pathway, and mutations of APC are observed in at least 70% of CRCs, especially in early colonic neoplasia [β [β-catenin pathway, its exact role in CRCs still needs more investigation.Wnts are a series of glycoproteins related to stimulation of receptor-mediated signal transduction pathways. The enrichment of nuclear  complex , 7, duri pathway . APC is eoplasia , 10. In lasia [β . Howeverin vivo or in vitro [β-catenin pathway was prevented by genistein, which reduced colon preneoplasia in rats [β-catenin signaling via a GSK-3β-independent pathway [With the increasing of old population , 13, thein vitro –17. Zhan in rats . Reabroi pathway . Aisha e pathway .Rubus idaeus L.) is a kind of economical berry crop that contains numerous potential healthy compounds, such as polyphenols, flavonoids, kaurane-type diterpenoids glycosides, oleanane, sodium-type triterpenoids, glycosides, and esters [Red raspberry  was mixed, homogenized by polyteron homogenizer, vacuum filtered, and evaporated at 45°C to extract the chemical extract of red raspberry. The main component was determined as raspberry ketone (4-p-hydroxyphenyl-2-butanone) by HPLC, ellagic acid, and polysaccharide. This method takes into account the effectiveness of chemical extracts of red raspberry plants and the safety of long-term human application after extraction and has been applied to the scientific research on the antihuman hepatocarcinoma effect of chemical extracts of red raspberry plants and published research papers [The 4-p-hydroxyphenyl-2-butanone (pure raspberry ketone) was purified by the School of Chemical Engineering, Harbin University of Technology. For natural hydroxyphenyl butanone, fresh red raspberry was purchased from Shangzhi City, Heilongjiang Province. In the preliminary study of our research group, 100 g red raspberry/300 ml absolute ethanol  was used firstly. Then, cell culture was performed by using 1640 medium (Gibco) supplemented with 10% serum  at 37°C-5% COμl 1640 medium, the medium was aspirated at 0 h, 24 h, 48 h, 72 h, and 96 h, and 100 μl of medium containing 10% CCK8 was added to each well. The OD value was read at a wavelength of 450 nm by a microplate reader for 4 hours. The 0 h OD value was used as the baseline, and the ratio of OD value to 0 h OD value at each time point was the cell proliferation multiplication. The cell proliferation multiple of the control group was base 1 at each time point, and the inhibition rate of each group was equal to one minus the proliferation ratio of each group/the proliferation ratio of the control group.One thousand and fifty hundred cells/well in 96-well plates were plated into cells. After adhering to 200 5 cells/well were plated into the cells, and 1.5 ml of 1640 medium containing 0.5% serum was added, and 2.6 ml of 1640 medium containing 10% serum was added to the lower chamber. The cells were photographed under a crystal violet staining microscope after incubation at 37°C-5% for 24 hours. Four field of view counts were randomly selected in each group.The cells were seeded at 400 cells/well in a 6-well plate and routinely cultured until each single cell clone was more than 50 cells, and crystal violet was fixed. The number of single cell clones was counted. Transwell plate  was evenly spread in the upper chamber, and 3 × 10The cells cultured in adherent cells were trypsinized without EDTA, and then, the cells were collected by centrifugation, fixed in 75% ethanol at 4°C for 24 hours. PI stained for 30 min, and the cell cycle was measured by flow cytometry. After adhering to the cells on the glass plate, 4% formaldehyde was fixed, 10% triton-X100 was permeated, 5% BSA was blocked for one hour, and 0.5% BSA diluted primary antibody  was incubated at 37°C for 2 hours; the secondary antibody diluted with 0.5% PBS was incubated at 37°C for 30 minutes, and the nuclei were counterstained with 0.1% diluted DAPI for 5 minutes. The antifluorescence quencher was mounted and photographed under a fluorescence microscope.6 cells/100 μl, electrophoresed on SDS-acrylamide gel, and blotted onto a 0.2 μm PVDF membrane. After 1 hour when 5% skim milk was sealed, 0.1% PBST was washed and then immersed in 0.1% PBST diluted primary antibody overnight; 0.1% PBST was washed, immersed in secondary antibody for 1 hour; 0.1% PBST was washed, and developer was added dropwise. Then, the ECL Western Blot Analysis System  was involved to perform the parallel grayscale analysis.The cells were counted who has the same number, and total protein was extracted by adding RIPA lysate at a concentration of 1 × 10siRNA (Suzhou Gemma Gene) sequence is as follows:Control group (NC)Justice chain (5′-3′): UUCUCCGAACGUGUCACGUTTAntisense strand (5′-3′): ACGUGACACGUUCGGAGAATTβ downregulation gene group (GSK3β-SI)GSK-3Justice chain (5′-3′): GGACUAUGUUCCGGAAACATTAntisense strand (5′-3′): UGUUUCCGGAACAUAGUCCTTμl of lipofectamin 2000, and 200 μl of Opti-MEM medium) was added to each well. Further experiments were carried out after incubation at 37°C-5% CO2 for 48 hours.Human colorectal cancer HCT116 and LOVO cells were evenly plated into 6-well plates and cultured for 24 hours. The cell density was about 50%. Premixed transfection solution .The adherent cultured cell samples were extracted with 1 GSK-3β gene primer sequence is as follows:Upstream 5′-3′: AGACGCTCCCTGTGATTTATGTDownstream 5′-3′: CCGATGGCAGATTCCAAAGGGAPDH primer sequence is as follows:″Upstream 5′-AGGCTGTGGGCAAGGTCATC-3Downstream 5′-TCAGGTCCACCACTGACACG-3′Fluorouracil -pyrimidine dione, 5-FU) is a classic antitumor drug for colorectal cancer. At present, most of the chemotherapy regimens for large intestine are centered on fluorouracil or drugs that can produce fluorouracil through in vivo transformation. And it is also the commonly used positive control drug in antitumor research of colorectal cancer [H-pyrimidThe proliferation inhibition effects of natural hydroxyphenyl butanone, raspberry ketone, and fluorouracil were detected by CCK8 method at different times and different concentrations in colorectal cancer HCT116 and LOVO cell lines. The optimal half-inhibitory concentration of the two colorectal cancer cell lines for 72 hours culturing was determined by gradient inhibition concentration experiments and calculated by the CompuSyn software .The calculated half-inhibitory concentrations and 1/2 and 1/4 half-inhibited concentrations of the different red raspberry plant chemical extracts, treated for 72 hours, were calculated as the high, medium, and low experimental group concentrations according to specific colorectal cancer cell line. The half inhibitory concentration of raspberry ketone and fluorouracil, treated for 72 hours, applied as the concentration of the positive control group .The cell proliferation of colorectal cancer HCT116, LOVO and normal human colonic epithelial cell line NCM460 was measured at the experimental concentration. The calculated inhibition rate was as follows: 48.60 ± 6.10%/9.62 ± 5.40% for the HCT116/NCM460 high-dose raspberry group, 21.78 ± 3.79%/−2.96 ± 5.50% for the middle dose raspberry group, and 12.15 ± 3.14%/−101.30 ± 12.28% for the low-dose raspberry group, while 53.65 ± 4.60%/21.45 ± 8.57% for the raspberry ketone group and 46.48 ± 8.59%/12.06 ± 6.19% in the fluorouracil group. For LOVO/NCM460 cell lines, the results were shown as the high-dose raspberry group 54.36 ± 2.65%/−1.48 ± 5.16%, medium-dose raspberry group 15.55 ± 3.75%/−8.31 ± 8.91%, low-dose raspberry group 6.11 ± 4.00%/−52.62 ± 10.36%, raspberry ketone group 50.39 ± 2.81%/16.37 ± 6.54%, and fluorouracil group 51.64 ± 6.92%/8.69 ± 5.19%. Our analysis results showed that both of the experimental group and the positive control group have an inhibitory effect on the proliferation of colorectal cancer cell lines, and the inhibitory effect is positively correlated with the concentration of the experimental group. The experimental group and the positive control group had higher inhibitory effect on the proliferation of colorectal cancer cell lines than on normal colon cell lines. The low and medium concentration experimental group could promote the proliferation of normal colon cell lines .After adding experimental concentration of red raspberry phytochemical extract and raspberry ketone positive control to the medium, the number of colony formation was as follows: HCT116 control group 27 ± 3.37, high-dose raspberry group 10.5 ± 2.38, medium-dose raspberry group 20.75 ± 2.22, low-dose raspberry group 27.75 ± 3.86, raspberry ketone group 11 ± 2.16, and fluorouracil group 12 ± 2.93. For LOVO cell lines are as follows: control group 34 ± 5.42, high-dose raspberry group 8.25 ± 0.96, medium-dose raspberry group 22.25 ± 1.50, low-dose raspberry group 29.75 ± 4.03, raspberry ketone group 11.25 ± 2.99, and fluorouracil group 12.25 ± 3.86. The colony forming ability of HCT116 and LOVO cells decreased in both of the experimental group and the positive control group, and the degree of decrease was positively correlated with the concentration of the experimental group. The colony forming ability of the low concentration experimental group was close to or equal to control group . ProlifeThe transwell experimental cell invasion rate was detected in colon cancer HCT116 and LOVO cells with red raspberry plant chemical extracts or raspberry ketone treatment. The results of transwell were shown as HCT116 control group 27.00 ± 3.67%, high-dose raspberry group 10.50 ± 2.38%, medium-dose raspberry group 20.75 ± 2.22%, low-dose raspberry group 27.75 ± 3.86%, raspberry ketone group 11.00 ± 2.16%, and fluorouracil group 12.00 ± 2.94%; and LOVO control group 91.75 ± 14.41%, high-dose raspberry group 23.75 ± 5.12%, medium-dose raspberry group 52.00 ± 5.71%, low-dose raspberry group 76.00 ± 8.40%, raspberry ketone group 24.25 ± 3.11%, and fluorouracil group 27.25 ± 3.96%. The lower cell invasion ability was detected in the experimental group and the positive control group, and the degree of weakening was positively correlated with the concentration of the experimental group. The cell invasion ability of the low concentration experimental group was close to or equal to that of the control group .β-catenin showed decreasing expression in the experimental group and positive control group after red raspberry phytochemical extract and raspberry ketone treatment, while β-catenin protein plays a regulatory role in the nucleus by binding to downstream-related nuclear factors. Furthermore, the distribution of β-catenin protein transferred from cytoplasm and nucleus in the control group became mainly in the cytoplasm, and the distribution in the nucleus was significantly reduced : G1 phase control group 41.54 ± 0.50%/38.31 ± 1.33%, high-dose raspberry group 57.78 ± 2.04%/59.22 ± 1.28%, medium-dose raspberry group 50.00 ± 0.59%/52.00 ± 2.00%, low-dose raspberry group 49.17 ± 1.34%/43.62 ± 1.63%, and raspberry ketone group 65.15 ± 0.91%/61.77 ± 1.11%; S-phase control group 51.07 ± 2.12%/50.70 ± 1.99%, high-dose raspberry 39.20 ± 1.58%/36.32 ± 1.35%, medium-dose raspberry group 46.43 ± 2.27%/43.00 ± 3.00%, low-dose raspberry group 48.66 ± 2.83%/53.85 ± 2.81%, and raspberry ketone group 23.56 ± 0.79%/30.64 ± 1.28%; and G2 control group 7.38 ± 1.70%/11.09 ± 1.58%, high-dose raspberry group 3.02 ± 0.54%/4.46 ± 1.12%, medium-dose raspberry group 2.00 ± 1.73%/5.00 ± 3.00%, low-dose raspberry group 2.17 ± 1.73%/2.53 ± 1.58%, and raspberry ketone group 11.30 ± 1.70%/7.64 ± 0.81%. The G1 phase cells in the experimental group and the positive control group increased, and the S phase cells decreased, suggesting that there is a G1-S phase cell cycle arrest; the degree of blockade is positively correlated with the concentration of the experimental group, and the concentration of the low concentration experimental group is close to or equivalent to the control group Figures .β-catenin, CyClinD1, and CDK4 protein were downregulated in Western blot, and GSK3β and APC protein expressions were upregulated, resulting that WNT channel as a whole is in a state of reduced function after treatment. Red raspberry phytochemical extracts can cause the downregulation of the WNT pathway key protein β-catenin in the colorectal cancer cell and the decrease in the distribution of nuclei in its function region. The downregulation of the function of the WNT pathway and cell cycle-related proteins CyClinD1 and CDK4 downstream of the WNT pathway leads to cell cycle arrest  of the control group in the colorectal cancer HCT116 and LOVO cell lines while the protein level expression is also significant downregulated (Figures β was downregulated by siRNA transfection, colorectal cancer HCT116 and LOVO cells were treated with red raspberry plant chemical extracts and raspberry ketones, WNT pathway protein constitutive proteins β-catenin, CyClinD1, and CDK4 on Western blot. The change from the control group is reduced (β could rescue the effect of red raspberry plant chemical extracts on proteins related to cell cycle regulation downstream of GSK-3β in the WNT pathway. After the expression of GSK-3β was downregulated by siRNA transfection, the experimental concentrations of red raspberry plant chemical extract and raspberry ketone positive control were added to the medium, and β-catenin protein was compared with the control group by qualitative analysis of immunofluorescence. The difference in expression level could be rescued, and the difference in expression distribution in the nucleus and cytoplasm is also reduced (The siRNA knockdown could reduce the GSK-3 Figures . After t reduced . The siR reduced . Our resβ-catenin plays a significant role in the inhibition of malignant expression of tumor cells through LEF/TCF and other pathways with its increase in expression and import to nucleus. This study showed that the phytochemical extracts of raspberry and raspberry ketone could downregulate the expression of Wnt pathway protein and reduce the nuclear import of β-catenin. The use of siRNA to blocking the Wnt pathway was significantly weakened, indicating that the phytochemical extracts of red raspberry plants and raspberry ketone exert an antitumor effect through the Wnt pathway in colorectal cancer cell lines (As an important signaling mechanism for cell growth and development, the Wnt pathway is closely related to tumors, especially colorectal cancer. As an important transcriptional regulator in the Wnt pathway, ll lines .Our group determined the mature method of phytochemical extracts of red raspberry plants through preliminary research and found that the phytochemical extracts of red raspberry plants can inhibit the malignant manifestations of human hepatocellular carcinoma through PTEN/AKT pathway . StudiesThis study showed that the phytochemical extracts of raspberry plants and raspberry ketone can cause cell cycle G1-S arresting of colorectal cancer HCT116 and LOVO cells through Wnt pathway, inhibiting the proliferation, colony formation, and invasion. The progression of the cell cycle is tightly regulated by a complex network of cyclins, CDKs, and transcription factors at several irreversible regulatory points. The cell cycle is regulated by the CyclinD1/CDK4 complex in the early G1 . CyclinDβ of the Wnt protein in the Wnt pathway was blocked for study.As a promoter of the Wnt signaling pathway, the Wnt family contains 14 proteins. This study found that red raspberry plant chemical extracts and raspberry ketones can cause downregulation of Wnt1, Wnt3a, and Wnt5a proteins, suggesting that it may have an inhibitory effect on multiple proteins of the Wnt family. Because the raspberry phytochemical extract and raspberry ketone may cause the downregulation of multiple Wnt family proteins, it is not suitable to verify the effect on the Wnt pathway by blocking a single Wnt family protein alone. In this study, the next level of the signal protein GSK-3Because we did not find an antitumor drug that is widely recognized and has the same antitumor mechanism as the chemical extract of the red raspberry plant, as a positive control, we chose a low concentration classic antitumor drug fluorouracil of colorectal cancer as a positive control for the cell phenotype to express the inhibition of chemical extracts of red raspberry plants on colorectal cancer cells. The mechanism of action of fluorouracil and red raspberry phytochemical extracts on colorectal cancer tumor cells was different in the cell cycle and Wnt pathway. It should not be used as a positive control. We only used pure raspberry ketone as a positive control.Currently, clinically widely used colorectal cancer antitumor drugs such as platinum, paclitaxel, and fluorouracil are toxic to normal cells while exerting a killing effect on tumor cells. In this experiment, when the half-inhibitory concentration of tumor cells was reached, the inhibitory effect of chemical extracts of red raspberry on normal colon cells was weaker than that of fluorouracil. Under the same experimental concentration, proliferation inhibition of colorectal cancer HCT116 and LOVO cell lines exerted by chemical extracts of raspberry plants and raspberry ketone was significantly stronger than that of normal colonic epithelial cells. The chemical extracts of red raspberry in the low-intensity experimental group promoted the proliferation of normal colonic epithelial cells, suggesting that the inhibitory effect of chemical extracts of red raspberry on colorectal cancer cells may be a selective antitumor effect, which may be less toxic or even have a certain cytoprotective effect on normal colon tissue.In the macrolevel, as a kind of widely used of raspberry fruit and functional food, its function mainly displays in the traditional medicine in the regulation of the body function, rather than the direct toxic effect of tumor; red raspberry has certain protective effect on normal colon tissue, which may be the macroeffect of microreaction in cell experiments. In terms of the microscopic molecular mechanism, this study found that red raspberry phytochemical extracts had inhibitory effects on Wnt pathway activity of colorectal cancer cells, and abnormal Wnt pathway as an important characteristic of colorectal cancer cells has been recognized as a result. There was no obvious abnormal activation of Wnt pathway in normal colon cells, which may be part of the reason why the chemical extract of red raspberry had weak toxicity and even certain protective effect on normal colon tissues. Of course, more detailed molecular mechanisms require a lot of further research is also one of the directions of further research.Extraction pure raspberry ketone is difficult and expensive, and there is no research on the safety of drugs in humans. The chemical extract of red raspberry plant can cause the inhibition of colorectal tumor cells similar to pure raspberry ketone, and as a widely consumed food extract, its safety in human application is fully guaranteed. This study shows that the chemical extract of red raspberry has broad application prospects as a functional food against colorectal cancer. Of course, the conversion to clinical applications requires a large number of animal and human trials.


Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated.Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in T. gondii.In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection.MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo.MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of The online version contains supplementary material available at 10.1186/s13071-021-04878-0. Toxoplasma gondii tachyzoites, as single-celled eukaryotes, undergo binary divisions, including karyokinesis and cytokinesis, in a process termed endodyogeny [odyogeny –3, whereodyogeny . The IMCodyogeny . At the odyogeny .TgIMC1 of immature parasites was reported to be highly soluble in detergents, whereas TgIMC1 of mature parasites was not. The conversion of IMC1 from a detergent-soluble network to a detergent-resistant network coincides with the proteolytic removal of the carboxyl terminus of TgIMC1. At later stages of daughter cell maturation, a 5 kDa peptide is removed from the C-terminus of IMC1 by a proteolytic process, allowing for the maturation of IMC1 with greater stability and mechanical strength to maintain cell shape and integrity [IMC formation undergoes a process that includes initiation, elongation, emergence and maturation, while some are synthesized de novo, using some components recycled from the maternal cells , 7. The ntegrity .Toxoplasma gondii expresses many cysteine proteases, such as cathepsin proteases, which are critical for the growth and survival of the parasite [T. gondii [Leishmania [T. gondii proliferation. To verify the hypothesis that metacaspases (MCAs) regulate the cell cycle of tachyzoites, we constructed an MCA2 knockout strain (Δmca2) and an MCA1/MCA2 double-knockout strain (Δmca1Δmca2) from Δku80 and demonstrated the requirement of MCA1 and MCA2 for IMC1 maturation, proliferation and virulence of T. gondii.parasite , 11. We . gondii . Metacas. gondii , 14. Met. gondii , 16. In . gondii , 18. Metishmania , 20. In ishmania , implyinToxoplasma gondii RHΔku80 tachyzoites were maintained in vitro by serial passages on confluent Vero cells at 37 °C with 5% CO2 in a humidified incubator. The TgMCA1 knockout strain (Δmca1) [T. gondii RHΔku80.Human foreskin fibroblast (HFF) and Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose and 4 mM glutamine supplemented with 10% fetal bovine serum  as described previously . Toxopla (Δmca1)  was incug for 10 min and washed in phosphate-buffered saline (PBS). The purified parasites were lysed in RIPA buffer  with the protease inhibitor PMSF . The lysate (7–10 µg) was subjected to SDS-PAGE (8% w/v for MCA1 and 6% w/v for MCA2) electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were sealed with 5% (w/v) skim milk in PBS and then incubated for 1 h at room temperature with mouse anti-rTgMCA1/2 antibody at 1:500 dilution in 5% skim milk in PBS, followed by goat anti-mouse IgG (H+L) horseradish peroxidase  as secondary antibody at 1:5000 dilution in 5% skim milk in PBS. Finally, appropriate chemiluminescent reagents  were used for reactive band visualization.Freshly isolated parasites were harvested and purified by filtration through a 5 µm filter, and were collected by centrifugation at 1400×2 for no more than 20 h and fixed for 15 min in 4% formaldehyde, after which they were permeabilized with 0.25% Triton X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for 30 min. Subsequently, the cells were incubated with primary antibody , mouse anti-TgIMC1 polyclonal antibody at 1:500 dilution, mouse anti-TgGAP45 polyclonal antibody at 1:500 dilution, mouse anti-TgGRA1 polyclonal antibody at 1:100 dilution) at 37 °C for 1 h, followed by FITC-conjugated goat-anti mouse IgG (H+L)  at 1:100 dilution with 3% BSA at 37 °C for 1 h. The nucleus was stained with Hoechst 33258  for 5 min. Rabbit anti-TgSAG1 antibody serum was used as a control. The parasites were observed and images were obtained using a Leica confocal microscope system . Images were processed using LAS AF Lite 2.2.0 software.Immunofluorescence assays (IFA) for TgMCA1 and TgMCA2 subcellular localization were performed as described previously . Prior tTgMCA2 were amplified from the Δku80 genome. PDHFR TgMCA2 KO was used to disrupt the native loci of the Δku80 tachyzoites by double homologous recombination and replacement of the entire coding region of TgMCA2, and stable clones were derived by pyrimethamine selection. The primers were designed to identify the correct clones, which were also confirmed by western blot. Meanwhile, the TgMCA2 complete coding sequence under the TgGRA1 promoter was transfected into Δku80 and the parasites were selected by pyrimethamine. To further characterize the role of TgMCAs, we generated a strain lacking both TgMCA1 and TgMCA2 (Δmca1Δmca2) based on Δmca1. The double-knockout parasites were screened under the pressure of pyrimethamine and chloramphenicol. Clones were identified by PCR and western blot as described above.The parental strain used to generate the knockout mutants was RHΔku80 (Δku80). Briefly, approximately 2000 bp of the 5′ flanking and 3′ flanking sequences of 2 for 7 days without disturbance. After 7 days, the medium was removed from the HFFs and the cells were washed five times with PBS. Cell monolayers were fixed with 4% formaldehyde for 10 min, stained with 0.2% crystal violet solution for 30 min, washed with deionized water, and then visualized by microscopy . The six-well plates were scanned using a Canon digital scanner .The plaque assay was performed on HFF cells cultured in six-well plates  as previously described . A total6) were inoculated on HFFs in 12-well plates. After 30 min, the extracellular parasites were removed by washing with PBS. After 24 h of incubation, infected cells were fixed with 4% formaldehyde, and parasites were stained with rabbit anti-T. gondii positive serum following the IFA protocol. The number of parasites per vacuole was counted using a fluorescence microscope  at ×400 magnification, and the number of tachyzoites per vacuole was scored with a total of 100 parasitophorous vacuoles (PV) for each strain. Data were analyzed by three independent experiments.Freshly isolated parasites  were inoculated onto HFFs in 12-well plates in cell culture medium at 37 °C with 5% CO2 for 2 h. The medium was then replaced with cyst induction medium and incubated at 37 °C without CO2 for 4 days. After fixation in 4% formaldehyde for 15 min, the cells were permeabilized with 0.25% Triton X-100 for 15 min and then blocked with 3% bovine serum albumin (BSA) for 30 min. Cells were then incubated with dolichos biflorus agglutinin (DBA), mouse anti-TgBAG1, or anti-TgGAP45 antibody diluted 1:50 at 37 °C for 1 h, as well as secondary antibody and Hoechst 33258. Coverslips were observed and images were obtained using a Leica confocal microscope system . Images were processed using LAS AF Lite 2.2.0 software, and the rate of bradyzoite formation was calculated by dividing the number of PVs of DBA-positive parasites by the total number of PVs of anti-TgGAP45 serum-labeled parasites. Data were obtained from three independent experiments, each in triplicate, and a total of 100 PVs were counted in each well.The cyst induction medium was prepared with 1.35% DMEM without sodium bicarbonate, 1 M HEPES, 5% serum and 1% antibiotics, and the pH was adjusted to 8.2 with KOH. The medium was stored at 4 °C before use. Freshly isolated parasites .t-test. In all cases, the two-tailed P-value was calculated and differences were considered significant if the P-value was ≤ 0.05.Statistical tests for virulence, intracellular parasite replication and the rate of cyst induction were performed using SAS . Two-way ANOVA was used to analyze the parasite replication data, and the chi-square was used to analyze each two parasite lines. The virulence of the strains to mice were determined used the life span test. The rate of bradyzoite induction in vitro was analyzed via the Toxoplasma Informatics Resource by BLAST using the amino acid sequences of metacaspases from Saccharomyces cerevisiae. They were named MCA1 (TGGT1_206490), MCA2 (TGGT1_278975) and MCA3 (TGGT1_243298) in our study. The transcriptional levels of MCA1, MCA2 and MCA3 are extremely low (see ToxoDB website), and the FPKM values in tachyzoites are 1/100th of the surface antigen protein 1 (SAG1) values and 1/1000th of the dense granule protein 1 (GRA1) values. However, the CRISPR Screen Value was −2.74 for MCA1, −3.93 for MCA2, and −2.97 for MCA3 , indicating that all three genes are functionally important to parasites.Three ICE family protease (caspase) P20 domain-containing proteins were identified in the ToxoDB TgMCA1 antiserum, we observed that MCA1 was distributed in the cytoplasm as discrete spots using an anti-HA monoclonal antibody  of the Δku80 strain and demonstrated that MCA1 is involved in the apoptotic pathway, but does not affect the growth and virulence of the parasites. To further evaluate the function of TgMCAs, we generated a MCA1/MCA2 double-knockout mutant. MCA2 in the Δmca1 mutant was replaced by DHFR (dihydrofolate reductase) and was screened by pyrimethamine. Replacement of the endogenous MCA2 locus was confirmed by genomic PCR using the indicated oligonucleotides  and BAG1 (related bradyzoite-specific surface protein) were detected in Δmca1Δmca2 , which indicated the success of the infection. Surprisingly, we detected TgBAG1 protein in the brain tissue of part of the surviving mice  was constructed to target complete TgMCA2 gene, and genomic PCR analysis of Δmca2 confirmed the absence of MCA2 gene. P1P2 and P3P4 were used to amplify 5′ and 3′ flanking sequence of TgMCA2, P5P6 was used to amplify parts of coding sequence of TgMCA2. b, c and d, PCR and western blot were performed to confirm TgMCA2 was completely knockout.Additional file 2: Figure S2. The localization of surface antigen (SAG1), actin and dense granule protein (GRA1) in double knockout strain. The IFA was performed to investigate whether the outer membrane and parasitophorous vacuoles of double knockout strain had altered. a–c showed there was no significant difference between Δku80 and Δmca1Δmca2 on SAG1, actin and GRA1. Loss of MCA1 and MCA2 did not influence the parasite outer membrane and PVs. The scale bar is 5 μm.