We expect that this work can provide theoretical guidelines for the design of novel porphyrin materials.Expanded porphyrin systems are capable of binding a variety of substrates due to their increased cavity size and aromatic nature, holding important applications as magnetic resonance imaging contrast agents and as sensitizers for photodynamic therapy. It is there of fundamental interest to know the photoelectrical properties of expanded porphyrins using quantum chemistry calculations. In this work, we theoretically designed and screened a series of expanded porphyrins by incorporating terthiophene (TTH) and dithienothiophene (DTT) moieties. Our calculations showed that all the designed molecules exhibit excellent optoelectronic performance than the reference molecule. It is suggested that the porphyrin molecule with TTH moiety has better stability than the one with DTT moiety. Finally, we demonstrated that molecule  Aromaticity is a fundamental and important concept in the field of chemistry with wide applications . Many stvia a methine carbon bridge. Expanded porphyrins exhibit structures, electronic properties, coordination chemistry, and reactivities that are entirely different from those of porphyrins. Expanded porphyrins exhibit intriguing Hückel and Mobius topologies than tetrapyrrolic porphyrin macrocycles in view of their larger-sized macrocyclic core  has been widely used as the π-conjugated building fragments to substitute pyrrole unit in porphyrin skeleton . Dithien1–8, In this work, we theoretically designed eight macrocycle molecules by incorporating TTH and DTT subunits, coded as All calculations are performed with DFT/TD-DFT methods, as implemented in the Gaussian 09 software . Geometr1–4) or a DTT unit (5–8). Molecules 1, 2, 5, and 6 feature the N heteroatom in the pyrrole group, whereas molecules 3, 4, 7 and 8 contain the S heteroatom in the pyrrole group. The key geometrical parameters of all molecules are listed in The ground state structures of the designed molecules are optimized at the B3LYP/6-311G  level of theory, as presented in 7-N8 bond lengths of molecules 1, 2, 5, and 6 are 7.058, 7.564, 6.106, and 6.172 Å, respectively. The S7-S8 bond lengths of molecules 3, 4, 7, and 8 are 6.520, 6.578, 5.175, and 5.394 Å, respectively. It is obviously that both bond lengths increase in the order of 7 < 8 < 5 < 6 < 3 < 4 < 1 < 2. For bond angles, C4-C5-C6 of molecules 1-8 is 125.5°, 125.6°, 129.2°, 129.7°, 119.8°, 120.8°, 123.2°, and 123.4°, respectively. The C9-C10-C11 bond angles of all molecules change following the same order as the C4-C5-C6 bond angles. The dihedral angles of S1-C4-C5-C6, N7-C9-C10-C11, and S7-C9-C10-C11 are listed in 1–4 are nearly zero, indicating that the four molecules would have better coplanarity. 1–4 has planner structure, whereas 5 and 6 exhibit bowl structure. For 7 and 8, the DTT unit is perpendicular to the porphyrin core, whereas the three thiophene units of the porphyrin core are nearly coplanar, as evidenced by the values of dihedral angles. The calculated ASE values are listed in 2 > 1 > 4 > 3 > 6 > 5 > 8 > 7, indicating that stability of molecules decreases in the same order.It can be seen that the NTo gain a deep understanding of the stability, the aromaticity of atomic rings of all molecules is calculated. Positive values of NICS indicate antiaromaticity. The more negative  NICS value suggests better stability. The critical points are analyzed at the geometrical center of a ring or above a molecular plane. To avoid the in-plane components, the NICS values are calculated at 1 Å above the thiophene and pyrrole rings , molecular center (point k), and intramolecular  using the flow around the gauge-independent atomic orbital method. An illustration of the different critical points is detailed in 1–8. The NICS (1) values at 1 Å above the critical points a, b, and c for 1–4, and critical points a and c for 5–8 are close. An exception is the NICS (1) value of 5–8 at 1 Å above the critical point of ring b show much smaller absolute values. It shows that the middle thiophene moiety in TTH has larger aromaticity than in DTT. In addition, the NICS (1) values of 1–8 at 1 Å above the critical points of rings d and f are close. The intramolecular 1 Å above critical points g, h, i, and j for 1–8 are similar. Moreover, comparisons of the NICS (1) values at critical point e for molecules with pyrrole N replaced by Se and S suggest that the inverted thiophene ring and selenophene ring are more stable than the conventional structure. As for the molecular center 1 Å above critical point, the NICS (1) values are −20.56, −21.21, −17.43, −18.12, −16.54, −16.91, −15.38, and −15.67 ppm for 1–8, respectively, 7 < 8 < 5 < 6< 3 < 4< 1 < 2. We note that the NICS (1) value of porphyrin is only -14.98 ppm  values, as listed in 4.98 ppm . This inH-L, are also used to evaluate the stability of compounds. The Frontier molecular orbital diagrams and the ΔH-L of molecules 1–8 are shown in 1 and 2, HOMO orbitals exhibit bonding characters, whereas LUMOs exhibit antibonding characters. In contrast to 1 and 2, the HOMO of 3 and 4 show antibonding character, while the LUMO exhibits bonding character. For 5 and 6, the HOMOs display antibonding characters in DTT, while the LUMOs display bonding characters in DTT. For 7 and 8, the HOMOs display antibonding character at the bottom thiophene ring, while the LUMOs display bonding character at the bottom thiophene ring. A larger HOMO-LUMO gap indicates a more stable structure. The HOMO-LUMO gaps of 1 (2.29 eV), 2 (2.30 eV), 3 (2.14 eV), 4 (2.17 eV), 5 (1.94 eV), 6 (1.95 eV), 7(1.60 eV) and 8 (1.64 eV) are listed in 1–8, molecules 1–4 have larger HOMO-LUMO gaps than 5–8. A positive correlation between NICS (1)  and HOMO-LUMO energy gaps can be observed, that is the larger HOMO-LUMO gaps would have the more negative NICS (1) value. A similar observation has been reported in our previous works [33-34].Energy levels of HOMO, LUMO, and their gap, Δ1 and 2 can be attributed to HOMO→LUMO + 1. For 3–8, the major transitions are assigned to HOMO→LUMO. It is clear that the absorptions are significantly redshifted and intensified with the substitution of the pyrrole N atom by Se atoms as compared to one substitution by S atoms. One can also observe that the absorptions are significantly redshifted and broadened when incorporating DTT moiety. For 2, the lowest-lying excitation is calculated at 707 nm, and the major transition of HOMO→LUMO has a maximum oscillator strength of 1.403.The TD-DFT method is used to calculate the vertical electronic excitations, as reported in 1–8 with TTH and DTT units incorporated in the porphyrins using DFT and TD-DFT calculations. The stability of molecules is directly related to aromaticity, which can be evaluated using the ΔH-L values and the NICS (1) values. The calculation results show that all the designed molecules show larger aromaticity and excellent photophysical properties. It is also revealed that the molecule with TTH unit is more stable than the one with the DTT unit, which can be ascribed to the coplanarity of the TTH unit. For the designed molecules, the one with an inverted thiophene ring or selenophene ring is more stable than the one with a thiophene ring or selenophene ring. Substitution of the pyrrole N atom by Se heteroatom results in a more stable structure than by S heteroatoms. All the absorption bands of the molecules are located in the visible region. The major absorptions are from either HOMO→LUMO + 1 or HOMO→LUMO transitions, which are beneficial for the intramolecular charge transfer process. Compared to the other molecules, compound 2 stands out because it shows unique properties, that is, larger HOMO-LUMO gap, better coplanarity, and stronger aromaticity. We expect these theoretical studies could pave the way for the future development of novel porphyrin molecules.We have investigated the aromaticity, stability, and photophysical properties of


Although surgery is considered the first-line treatment for patients with endogenous Cushing’s syndrome (CS), medical therapy is often required to control severe hypercortisolism. Metyrapone and osilodrostat are both steroidogenic inhibitors targeting the 11β-hydroxylase, however, their therapeutic effectiveness has not yet been directly compared. This study aimed to evaluate metyrapone and osilodrostat in the short-term therapy of CS.Retrospective analysis of patients with endogenous CS treated with metyrapone or osilodrostat as monotherapy for at least 4 weeks. Main outcome measures were serum cortisol and 24h urinary free cortisol (UFC) at baseline (T0) and after 2 (T1), 4 (T2), and 12 weeks (T3) of therapy.vs osilodrostat: 817 µg/24h; p=0.93). From T0 to T1, the decrease of UFC was less pronounced under metyrapone than osilodrostat . This tendency persisted at T2  while at T3 a decrease in UFC from T0 was more pronounced in the metyrapone group . Under osilodrostat, a QTc-interval prolongation was identified at T3 (mean 432 ms vs 455 ms). From T0 to T2, the number of antihypertensive drugs remained comparable under metyrapone and decreased under osilodrostat (n= -0.3 vs n= -1.0).16 patients with endogenous CS were identified . Each 8 patients were treated with metyrapone and osilodrostat. Despite heterogeneity, both groups showed comparable mean UFC levels at T0 (metyrapone: 758 µg/24h Although both drugs show comparable therapeutic efficacy, osilodrostat seems to reduce cortisol levels and to control blood pressure faster. Endogenous Cushing’s syndrome (CS) is a rare disorder with an incidence of 0.2–5.0 per million people per year . If the Surgery is considered the first-line treatment for patients with endogenous CS , e.g. trThe primary aim of this retrospective monocentric study was to compare the short-term efficacy of metyrapone and osilodrostat on cortisol levels in patients with endogenous CS.Patients with endogenous CS admitted to the University Hospital Würzburg were retrospectively reviewed. Those who were treated with metyrapone or osilodrostat as monotherapy for at least four weeks between December 2017 and December 2021 were considered eligible. CS was diagnosed according to established criteria , 19. TheAll patients provided written informed consent to at least one of two disease-specific clinical registries, which were approved by the local ethics committee of the University Hospital of Würzburg .As previously performed , 21, comFor the analysis of the QTc-interval the Bazzett formula [QTc=QT/√(RR/1seconds)] was used.p-value < 0.05 was considered statistically significant.Continuous variables were reported as mean ± standard error of mean (SEM) or as median with range, whereas categorical variables were provided as numbers and percentages. Data distribution was evaluated with the Shapiro-Wilk test. Parametric and non-parametric data were analyzed with Student´s T-tests and Mann-Whitney U test, as appropriate. Dichotomic variables were analyzed with the Fisher’s exact test or the Chi-square (χ2) test. To compare the effect of metyrapone and osilodrostat on hormonal and biochemical parameters, a two-way repeated measures ANOVA was used. The delta (change) percentage from T0 to a subsequent study time point was calculated to evaluate the alteration of a parameter during the course of medical treatment with metyrapone or osilodrostat. A Statistical Analysis was performed with SPSS version 26  and GraphPad Prism version 8 .In total, 7 patients with CD, 5 patients with ECS, and each 2 patients with CPA and ACC were analyzed. The metyrapone population consisted of 2 patients with CD, 4 patients with ECS, 1 patient with CPA, and 1 with ACC. The osilodrostat population included 5 patients with CD, 1 patient with ECS, 1 patient with CPA, and 1 patient with ACC Table 1.p < 0.0001).Mean time of therapy was 17.0 ± 3.4 weeks for the metyrapone group and 9.5± 1.1 weeks in the osilodrostat group , 1250 mg at T2 (n = 8), and 1250 mg at T3 (n = 5). For osilodrostat, median dose was 4 mg at T1 (n = 6), 6 mg at T2 (n = 8), and 7 mg at T3 (n = 4).p = 0.61 compared with T0), 22.3 ± 2.0 µg/dL at T2 (p = 0.67), and 8.3 ± 2.5 µg/dL at T3 (p = 0.007) (p = 0.99 compared with T0), 18.7 ± 4.3 µg/dL at T2 (p = 0.82) and 13.0 ± 1.6 µg/dL at T3 (p = 0.44) . During follow-up, mean serum cortisol was 21.0 ± 3.8 µg/dL at T1 (p = 0.63) (p = 0.57). At T2, serum cortisol levels were below 25 µg/dL in 5/8 (62.5%) patients under metyrapone and in 5/8 (62.5%) patients under osilodrostat. At T3, a more pronounced decrease of cortisol was found in the metyrapone than in the osilodrostat group , 281 ± 87 µg/d (p = 0.73) and 53 ± 25 µg/d (p = 0.62). On the other hand, mean UFC under osilodrostat at T0 was 817 ± 644 µg/d. During follow-up, UFC levels were 74 ± 36 µg/d at T1 (p = 0.44), 117 ± 34 µg/d at T2 (p = 0.41) and 131 ± 55 µg/d at T3 (p = 0.55)  . At this time point, 0/6 patients treated with metyrapone and 3/7 patients (42.9%) under osilodrostat had a normalized UFC. At T3, the delta change of UFC from baseline was more pronounced in the metyrapone group   in the metyrapone group was 758 ± 309 µg/d at T0. At T1, T2 and T3 was 748 ± 434 µg/d , whereas in the metyrapone group the systolic pressure did not change relevantly and the diastolic slightly increased   because of adverse events (asthenia and dizziness). 1 patient under metyrapone was lost to follow-up before T3. In the osilodrostat group, the therapy was discontinued in 1 patient with CD at T2 because of adverse events , and in 2 additional patients after tumor resection (1 CD and 1 ECS). Another patient under osilodrostat did not show up at T3 for unknown reasons and was lost to follow-up.p = 0.99), while in the osilodrostat group an increase  was detected.At T1, 1 patient under metyrapone and one under osilodrostat required potassium replacement therapy. At T2 and T3, two patients under metyrapone and 3 patients under osilodrostat required potassium replacement therapy. Analysis of potassium levels was performed only in patients without potassium replacement therapy. No significant differences in potassium levels were identified at T1 and T2. However, at T3, no substantial changes in potassium levels was identified in patients under metyrapone  and alanine aminotransferase (ALT), no substantial differences between the metyrapone and osilodrostat group were observed at T1, T2 and T3  was implemented. This approach might have underestimated the adverse events. Third, both groups were inhomogeneous in terms of CS subtypes and clinical characteristics .Nevertheless, to our knowledge, this is the first study that directly compared metyrapone and osilodrostat as short-term therapy of endogenous CS. The present analysis is, therefore, relevant for daily clinical practice, facilitating the choice of a certain steroidogenesis inhibitor when a prompt decrease of cortisol levels is indicated. Osilodrostat might be superior in rapidly reducing cortisol excess, but also clinical parameters like blood pressure. However, careful monitoring of the QTc intervals is required.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University Hospital of Würzburg . The patients provided their written informed consent to participate in this study.MD and BA designed the research. MD, BA and UD performed the statistical analyses and drafted the manuscript. All authors collected samples and clinical data from patients, contributed to writing the manuscript, and approved the final version to be published.This work was supported by the DFG German Research Foundation Project 314061271-TRR 205 (to MF) and the European Reference Network on Rare Endocrine Conditions (Endo-ERN). This publication was supported by the Open Access Publication Fund of the University of Würzburg.UD received honoraria from Recordati Rare Diseases for scientific board activities. TD received honoraria from Recordati Rare Diseases and HRA Pharma for scientific board activities.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


U-286 and U-548, to inhibit inflammation induced by ATP/LPS in RAW 264.7 cells via P2X7 receptors was demonstrated. It has been established that the selected compounds were able to inhibit ATP-induced calcium influx and the production of reactive oxygen species, and they also exhibited pronounced antioxidant activity in mouse brain homogenate. In addition, compounds U-286 and U-548 decreased the LPS-induced activity of the COX-2 enzyme, the release of pro-inflammatory cytokines TNF-α and IL-1β in RAW 264.7 cells, and significantly protected macrophage cells against the toxic effects of ATP and LPS. This study highlights the use of 1,4-naphthoquinones as promising purinergic P2X7 receptor antagonists with anti-inflammatory activity. Based on the data obtained, studied synthetic 1,4-NQs can be considered as potential scaffolds for the development of new anti-inflammatory and analgesic drugs.P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and ELISA approaches, the ability of 1,4-naphthoquinone thioglucoside derivatives, compounds  ATP is an important intercellular messenger and neurotransmitter that activates ligand-gated ion channels of the P2X family (P2XR) and metabotropic receptors of the P2Y family . These rP2X7R is expressed by hematopoietic immune cells, including dendritic and mast cells, T-cells, macrophages, and microglia . Long-teSo far, many non-selective P2X7R blockers have been discovered, such as BBG, PPADS, TNP-ATP, and others . CurrentInflammation is one of the major problems associated with diseases caused by P2X7R overactivation. However, there are no anti-inflammatory drugs on the pharmaceutical market based on selective blockers of these receptors . Modern Natural 1,4-naphthoquinones  are a promising class of biologically active compounds that perform various protective functions in plant and animal organisms . Most ofU-548 and U-557, and their tetracyclic thioglucoside derivatives, U-286 and U-556 were selected. An in vitro biological activity assay indicated that the selected compounds inhibited P2X7R functions in the Neuro-2a neuronal cells, suggesting their analgesic properties. The analysis of these compounds in silico indicated their potential binding to the allosteric site of P2X7R pyrano-oxathiine-6,11-dione ( plates) . 1H NMR U-286 and U-548 were synthesized according to the methodology described in [2O to the desired concentration.Compounds ribed in . Compoun4 cells/well) were seeded and incubated in 96-well plates for 24 h at 37 ℃, 5% CO2. 1,4-NQs were added to cells at final concentrations of 0.35–100.0 μM, using double dilutions. Plates were incubated for an additional 24 h. After incubation, the medium with tested compounds was replaced with 100 μL of pure medium. Cell viability was determined using the MTT -2,5-diphenyltetrazolium bromide) method, according to the manufacturer’s instructions . For this purpose, 10 μL of MTT stock solution (5 mg/mL) was added to each well and the microplate was incubated for 4 h at 37 ℃. After that, 100 μL of SDS-HCl (1 g SDS/10 mL dH2O/17 μL 6 N HCl) were added to each well followed by incubation for 4–18 h. The absorbance of the converted dye formazan was measured using PHERAstar FS plate reader  at a wavelength of 570 nm. The results were presented as percent of control data, and concentration required for 50% inhibition of cell viability (IC50) was calculated.RAW 264.7 cells (2 × 10All tested compounds were added to the wells of the plates in a volume of 20 μL dissolved in PBS (DMSO concentration ≤ 1%).4 cells per well) in a DMEM cultural medium and incubated overnight at 37 ℃, 5% CO2. Then, the cells were washed once with HBSS saline  and loaded with 5 μM Fluo-8 AM  and 0.05% (w/v) Pluoronic ® F-127  in the same buffer solution. Cells were incubated for 40 min  and then were washed with HBSS buffer without the fluorescent dye and treated by studied compounds during 20 min at RT in the dark. Standard inhibitors, non-selective P2X7R antagonist BBG (10 μM)  and competitive P2X7R antagonist A438079  were used as inhibitory controls. Fluorescence was measured with PHERAstar FS plate reader  by recording excitation signals at 490 nm and emission signals at 510 nm. ATP  was added using a robotic microinjector after the baseline recording. RAW 264.7 cells were seeded in 96-well plates . Further, the studied compounds were added to the cells at concentrations of 5, 1, and 0.1 μM. Standard P2X receptor blockers BBG and A438079 at a concentration of 10 μM were used as a comparison control. The fluorescence level was measured with a PHERAstar FS plate reader  at λex = 480 nm and λem = 520 nm, ATP 2 mM  was added after baseline recording and HBSS (20 μL) was added as a negative control. RAW 264.7 cells were seeded in 96-well plates at a density of 4 × 102 incubator at 37 ℃ and 5% CO2.Mouse macrophage cell line RAW 264.7 TIB-71™ was purchased from ATCC . Cells were grown in DMEM medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  in a CO4 cells/well) were seeded and incubated in 96-well plates for 24 h at 37 ℃ and 5% CO2 in an incubator. Compounds U-286 and U-548 were added to the wells at final concentrations of 5, 1, and 0.1 μM, and the plates were incubated for an additional 1 h. BBG (10 μM), A438079 (10 μM) and apyrase  were also added to cells as standard blockers of ATP action. Then ATP  and LPS  were added to the cells. Cells were incubated for 24 h, after which cell viability was determined using the MTT method, as described above.RAW 264.7 cells (4 × 104 cells per well of a 96-well plate) were seeded and incubated for 24 h at 37 ℃, 5% CO2 in an incubator. Then, the cells were treated with the studied 1,4-naphthoquinones at final concentrations of 5, 1 and 0.1 µM and incubated for 1 h. BBG (10 μM), A438079 (10 μM) and apyrase (30 U/mL) were used as inhibition control. Next, LPS  was added to the cells and the cells were incubated for 4 h. Thereafter, ATP  was added to the cells and the plates were incubated for an additional 24 h. Cells incubated without ATP and LPS were used as negative controls. The plate was then centrifuged at 250× g and 100 μL of the supernatant from each well was transferred to the relevant wells of an optically clean 96-well plate. An equal volume of the reaction mixture (100 μL) from the LDH Cytotoxicity Assay Kit  was added to each well and incubated for 30 min at room temperature. The absorbance was measured at λ = 490 nm using a Multiscan FC spectrophotometer . RAW 264.7 cells (4 × 10ex = 485 nm and λem = 518 nm. Cell viability is expressed as a percentage of control.A stock solution of fluorescein diacetate (FDA)  in DMSO (1 mg/mL) was prepared. RAW 264.7 cells were seeded and treated as described above. Further, FDA solution (50 μg/mL) was added to each well and the plate was incubated in the dark at 37 ℃ for 15 min. Cells were washed and fluorescence was measured using a Fluoroscan plate reader  at λ5 cells/mL) and incubated in DMEM in the presence of LPS  for 3 h. The cells were then washed twice and the cell medium was changed to PBS. Then, compounds U-286 and U-548 were added at concentrations of 1 and 0.1 μM, and the cells were incubated for 30 min. The standard P2X receptor blockers BBG (10 µM) and A438079 were used as inhibitory controls. After incubation with blockers, 1 mM ATP  was added and the cells were incubated for an additional 30 min. Cells incubated without LPS and ATP, or with only ATP, or with only LPS were used as controls. Next, the cells were washed with cold PBS  and lysed with RIPA buffer . The protein concentration in the samples was measured by the Bradford method. Proteins were separated by electrophoresis with sodium dodecyl sulfate (SDS) in 12% polyacrylamide gel. The separated proteins were then transferred to an Immobilon®-P polyvinylidene difluoride (PVDF) membrane  using a semi-dry Trans-blot® SD transfer cell . IL-1β protein zones were detected using specific primary polyclonal antibodies against IL-1β  at a dilution of 1:1000. Secondary antibodies were conjugated to horseradish peroxidase . β-actin zones were detected with specific monoclonal antibodies  applied at a 1:10,000 dilution and used as a loading control. The protein zones on the membranes were developed using the PierceTM ECL kit  according to the manufacturer’s instructions and visualized using the VersaDoc imaging system . Protein zone densitometry was assessed using Image Lab 6.0.1 .The expression of IL-1β was determined by Western blotting. To do this, RAW 264.7 cells were seeded in six-well plates (1.0 × 104/well) for 2 h at 37 ℃ in a 5% CO2 incubator. The cells were incubated 2 h after complete adhesion with U-286 and U-548 compounds at concentrations of 1.0 and 0.1 μM for 1 h. Then, LPS was added at a concentration of 1.0 μg/mL to each well and incubated for 24 h. Cells incubated without LPS and compounds or with LPS alone were used as positive and negative controls. To prepare a cell lysate, cells were washed once with 0.2 ml cold PBS, resuspended in 0.2 mL PBS, and centrifuged at 500× g for 3 min. The cell pellet was then resuspended in 0.1 mL lysis buffer with a mixture of protease inhibitors, shaken and incubated on ice for 5 min and centrifuged . The collected supernatant was stored on ice for later use. COX-2 activity was determined using a cyclooxygenase activity assay kit  according to the manufacturer’s protocol.RAW 264.7 cells were seeded in 96-well plates (2.0 × 104 per well) and incubated for 2 h  until complete adhesion. Then compounds U-286 and U-548 were added to the cells at concentrations of 1 and 0.1 μM. Cells were incubated with substances for 1 h, and then LPS (1.0 μg/mL) was added and incubated for 24 h. Cells incubated without LPS and compounds or with LPS alone were used as positive and negative controls, respectively. Samples were then centrifuged , and supernatants were collected and stored on ice for later use. Cells were washed gently with cold PBS and then resuspended at 1500× g for 10 min at 2–8°C to remove cell debris. The TNF-α level in the mixture of supernatants and cell lysates was immediately analyzed using the Mouse TNF-α ELISA kit , according to the manufacturer’s instructions.RAW 264.7 cells were seeded in 96-well plates (2 × 104 cells per well for 24 h. The medium was replaced with fresh DMEM and treated with LPS (100 ng/mL) for 3 h at 37 ℃. After that, the cells were treated with studied compounds  for an additional 30 min. Standard P2X7R inhibitor A438079 (10 µM) was used as inhibitory control. Then ATP  was added to each well for 30 min. For fluorescence measurement, the cells were washed once and cell medium was replaced by HBSS, containing 10 µm of fluorescent dye 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) . Cells were incubated for 30 min at 37 ℃, washed twice with HBSS without fluorescent probe and additionally incubated for 20 min at RT in the dark. The measurement of fluorescence intensity was carried out using PHERAstar FS plate reader  at λex = 485 nm and λem = 520 nm.RAW 264.7 cells were seeded in 96-well plates at a density 2 × 102HPO4, pH 7.4) and then homogenized using a Potter–Elehjem tissue homogenizer, adding the buffer to the brain in a weight ratio of 1:4. 10 mL of buffer was added to the resulting homogenate and centrifuged for 10 min at 900 rpm in a Labofuge 44R centrifuge . The supernatant was transferred to a volumetric flask and adjusted to 25 mL with a buffer. The Lowry protein concentration in homogenate was in the range of 0.27–0.3 mg/mL.The procedure was performed according to the protocol described in . The bra4 solution was added, and the reaction was stopped by adding of 20% trichloroacetic acid (TCA) . The homogenate was centrifuged, supernatant was collected and 0.7% solution of thiobarbituric acid (TBA)  in 50% glacial acetic acid was added to reveal the products reacting with TBA. The content of TBA-reacting substanses (TBARS) was determined spectrophotometrically using PHERAstar FS plate reader  by measurement of fluorescence intensity at 480/520 nm (λex/λem).To determine the antioxidant activity, the solution of the tested compounds was added to the brain homogenate, then 2 mM FeSO50, which is the concentration of compound that scavenged 50% of DPPH radicals in the reaction solution.DPPH radical scavenging activity of compounds was tested as described  with minAll data were obtained in three independent replicates and calculated values were expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) with post-hoc Student–Newman–Keuls test was performed using SigmaPlot 14.0 . A significant difference between two groups was considered achieved when p was < 0.05.


G6PD and PKLRR41Q variants.Glucose 6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PKLR) deficiencies are common causes of erythrocyte haemolysis in the presence of antimalarial drugs such as primaquine and tafenoquine. The present study aimed to elucidate such an association by thoroughly investigating the haematological indices in malaria patients with G6PD deficiency variants. The multivariate analysis was performed to investigate the association between anaemia and G6PD MahidolG487A, the most common mutation in this study.Blood samples from 255 malaria patients from Thailand, Myanmar, Laos, and Cambodia were collected to determine haematological profile, G6PD enzyme activity and G6PD MahidolG487A and PKLRR41Q variants were 7.1% and 2.6%, respectively. Compared with patients with wildtype G6PD after controlling for haemoglobinopathies, G6PD-deficient patients with hemizygous and homozygous G6PD MahidolG487A exhibited anaemia with low levels of haemoglobin . These patients also exhibited high levels of reticulocytes (3.60%). The median value of G6PD activity before treatment (Day 0) was significantly lower than that of after treatment (Day 28) . Reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0 . PKLRR41Q had no correlation with anaemia in malaria patients. The risk of anaemia inpatients with G6PDMahidolG487A was higher than wildtype patients . Univariate and multivariate analyses confirmed that G6PDMahidolG487A independently associated with anaemia (< 11 g/dl) after adjusted by age, gender, Plasmodium species, parasite density, PKLRR41Q, and haemoglobinopathies (p < 0.001).The prevalence of G6PD deficiency was 11.1% (27/244) in males and 9.1% (1/11) in female. The MAFs of the G6PD MahidolG487A, but not with PKLRR41Q, had anaemia during infection. As a compensatory response to haemolytic anaemia after malaria infection, these patients generated more reticulocytes. The findings emphasize the effect of host genetic background on haemolytic anaemia and the importance of screening patients for erythrocyte enzymopathies and related mutations prior to anti-malarial therapy.This study revealed that malaria patients with The online version contains supplementary material available at 10.1186/s12936-022-04267-7. G6PD gene on chromosome X. Genetically, males are either G6PD deficient or G6PD normal, while females can be homozygous with G6PD deficiency (mutations are present on both X chromosomes) or heterozygous (one X chromosome is affected) or G6PD normal. The frequency of G6PD status follows the Hardy Weinberg equilibrium. This makes heterozygous females are more common than hemizygous males and homozygous females are the least common [Glucose 6-phosphate dehydrogenase G6PD; EC 1.1.1.49) and pyruvate kinase PKLR; EC:2.7.1.40) deficiencies are the most common hereditary metabolic disorders affecting red blood cells [.1.1.49 a.7.1.40 dG6PD mutations, most of which are point mutations, have been documented [G6PD MahidolG487A, a common Southeast Asian mutation, has been reported to reduce Plasmodium vivax density [P. vivax clinical disease [Plasmodium spp., G6PD-deficient patients are susceptible to haemolytic anaemia when exposed to active and toxic metabolites of primaquine (PQ) and tafenoquine (TQ) [Plasmodium falciparum gametocytes for transmission and preventing the relapse of P. vivax and Plasmodium ovale malaria [G6PD in malaria patients prior to PQ and TQ administration are necessary to prevent adverse outcomes [Approximately 186 cumented . Each mucumented . Recentl density . Additioine (TQ) . PQ and  malaria . In 2013 malaria . The diaPK predominantly expressed in the liver and red blood cells (PKLR). More than 150 mutations of PKLR have been reported [G6PD and PKLRR41Q mutations have not been thoroughly investigated. The main aim of the present study was to examine the haematological profiles in malaria patients with G6PD or PKLR mutations.PK deficiency (PKD), the second most common enzyme deficiency, causes haemolytic anaemia worldwide with an estimated prevalence of 0.005% 1/20,000) in the Caucasian population. The prevalence of PKD in the European population was estimated to be less than 0.05%  /20,000 i, 12. Thereported . Evidencreported . RecentlPlasmodium spp. and had no history of anti-malarial drug treatment 2 weeks prior were recruited for this study. Blood samples were collected at the Hospital for Tropical Diseases in Bangkok, Thailand and transferred on ice to a research laboratory at the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand within an hour for immediate measurement of G6PD activity and haematological parameters. Complete blood count (CBC) was measured using an BC-6800 Auto Hematology Analyzer .The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University  . All patients were screened by passive case detection (PCD) protocol and provided written informed consent prior to enrollment in this study. A total of 255 uncomplicated malaria patients who were admitted to the Hospital for Tropical Diseases in Thailand during 2011–2012 with blood slide positivity for Plasmodium spp. were identified under a microscope by an independent parasitologist at the Hospital for Tropical Diseases and further confirmed by polymerase chain reaction (PCR)-based analysis [Giemsa staining of thick and thin blood smears prepared from finger pricks was examined every 12 h from initiation of treatment until they were negative. Blood smears were examined daily until patients were discharged. analysis .G6PD activity in the blood of malaria patients was measured in triplicate along with the normal and G6PD-deficient controls  using a quantitative assay kit for G6PD  prior to treatment and repeated weekly until patients were discharged. This assay measured NADPH production by G6PD in the blood of patients in parallel with positive and negative controls. Detection was carried out at a wavelength of 340 nm. The haemoglobin level was measured using Hb201  and used to calculate G6PD activity. Leftover blood samples were kept at − 20 °C for molecular typing.G6PD MahidolG487A, G6PD ViangchanG871A, and G6PD KaipingG1388A, as described previously [and PKLRR41Q according to a previous report [PKLRR41Q was amplified by PKLR-R41Q-F: 5'–GCC AAC GGG GTA TCT ACG GC–3' and PKLR-R41Q-R: 5'–GCA GAG GTG TTC CAG GAA GG–3'. After digestion with AciI, the PCR product size of PKLRR41Q was 121 base pairs (bp). The normal allele produced two fragments of 102 bp and 19 bp. For G6PD-deficient patients with unknown mutations by the PCR–RFLP method, the coding exons of G6PD (exons 3–12) were amplified using primers described previously [G6PD reference sequence (GenBank accession number X55448.1).Genomic DNA from frozen blood samples was extracted using the phenol–chloroform method. PCR–restriction fragment length polymorphism (PCR–RFLP) was used to identify eviously , and PKLs report . PKLRR41eviously . The PCR-α3.7, -α4.2, –SEA and –FIL variants of the α-globin gene. PCR–RFLP was used to identify HbCS and HbE, as described previously [For thalassaemia mutations, multiplex gap-polymerase chain reaction was performed as previously described with minor modifications  to deteceviously , 20.t-test was used to analyse the differences in quantitative variables. Haemoglobin less than 11 g/dl is considered anaemia [G6PD MahidolG487A/G6PD wildtype, a multiple linear regression was performed adjusted for age, gender, parasite species, parasite density, and haemoglobinopathies. A statistically significant difference was defined as two-sided with a p-value less than 0.05.All statistical analyses were performed using SPSS version 22 . Data are expressed as percentages, median ± interquartile range (IQR), and mean ± standard deviation (SD). Following a computation approach reported previously, the adjusted male median (AMM) of G6PD activity was used to determine the cut-off values for G6PD deficiency . The AMM anaemia . The risP. falciparum and P. vivax were 106 (41.6%) and 145 (56.9%), respectively. Three patients (1.2%) had coinfection of P. falciparum and P. vivax. One patient (0.4%) was infected with Plasmodium malariae.A total of malaria patients consisting of 189 individuals from Myanmar (74.1%), 35 Thais (13.7%), 16 Karen (6.3%), 8 Cambodians (3.1%), 4 Mons (1.6%), 2 Laotians (0.8%), and 1 unknown ethnicity (0.4%) are summarized in Table P. falciparum, 25 patients (55.6%) were infected with P. vivax, and 1 patient (2.2%) was coinfected with P. falciparum and P. vivax. Of all 28 patients with G6PD deficiency, 18 patients carried G6PD MahidolG487A , 3 patients carried G6PD ViangchanG871A , 1 patient carried G6PD KaipingG1388A  and 1 patient carried G6PD AuresT143C   of G6PD MahidolG487A was 7.1% in these populations (Table G6PD MahidolG487A and 3 hemizygous G6PD ViangchanG871A were 0.40 ± 0.46 U/g Hb (range from 0.00 to 0.89 U/g Hb) and 0.63 U/g Hb (range from 0.31 to 0.66 U/g Hb), respectively. G6PD activity levels in 1 hemizygous male with G6PD KaipingG1388A, 1 hemizygous male with G6PD AuresT143C, and 1 homozygous female with G6PD MahidolG487A were 1.02 U/g Hb, 0.40 U/g Hb, and 1.04 U/g Hb, respectively.The overall median value of G6PD activity in this cohort n = 255) was 5.66 ± 2.55 U/g Hb (median ± IQR), ranging from 0.00 to 14.59 U/g Hb. The median values of G6PD activity in males (n = 244) and in females (n = 11) were 5.68 ± 2.49 and 4.80 ± 3.10 U/g Hb, ranging from 0.00 to 14.59 U/g Hb and 1.04 to 7.95 U/g Hb, respectively. The adjusted male median (AMM) G6PD activity in G6PD normal was 5.77 U/g Hb. The cut-off values for G6PD deficiency and G6PD intermediate were < 1.73 U/g Hb (< 30% of the AMM) and 4.04 U/g Hb (30–70% of the AMM), respectively . These patients were from Thailand and Thailand borders , 2.6% in individuals from Myanmar (10 of 378), 2.9% in Thais (2 of 70) and 6.3% in Cambodians (1 of 16).The p = 0.003). These patients also had a significant increase in the reticulocyte count . Malaria patients with G6PD MahidolG487A mutation (n = 17) compared to wildtype patients without common Southeast Asian (SEA) mutations including the G6PD MahidolG487A (n = 215) exhibited a significant decrease in haemoglobin levels . These patients also had an increase of reticulocyte levels  , compared to malaria patients with normal G6PD activity levels (n = 209), exhibited a significant decrease in the haemoglobin levels  . The median value of G6PD activity in G6PD deficiency group on Day 28 was significantly different from that of on Day 0 . Additionally, reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0  (mean ± SD) . However, there were not statistically significant in reticulocyte count between Day 0 and Day 28 in G6PD deficiency group.In the longitudinal monitoring of G6PD activity before (Day 0) and after (Day 28) treatment, there was a total of 174 malaria patients with complete haematological data were analysed. These patients included G6PD deficiency, thalassaemia, and haemoglobinopathies. The median value of G6PD activity on Day 28 was significantly higher than that of on Day 0  Fig. a. In G6Pp < 0.001). Besides, mild to moderate anaemia was observed in 41.2% (7 of 17 cases) of patients with G6PD MahidolG487A and 16.7% (36 of 215 cases) of patient with wildtype G6PD (non-common SEA mutation) . The univariate analysis revealed that haemoglobin levels were significantly associated with both G6PD deficiency/G6PD normal (p < 0.001) and G6PD MahidolG487A/ G6PD Wildtype (p < 0.001)  and G6PD MahidolG487A/ G6PD Wildtype (p < 0.001) , moderate anaemia (Hb < 8 g/dl), and severe anaemia (Hb < 5 g/dl) . Mild toP. vivax infections. It also reduces transmission of P. falciparum. However, PQ and TQ cause acute haemolytic complications in patients with G6PD deficiency. Although there have been many reports of G6PD deficiency status and its mutations in malaria patients living in the Southeast Asia, data on the haematological parameters of G6PD and other erythrocytic mutations including PK in malaria patients are limited. These data may be beneficial for the administration of anti-malarial treatment especially PQ and TQ prescription.Malaria infection causes haemolysis of infected erythrocytes. PQ and TQ, 8-aminoquinoline, are essential anti-malarial drugs commonly used for radical cure of P. vivax infection was slightly higher than that of with P. falciparum infection, confirming that the frequencies of P. vivax and P. falciparum-infected cases are approximately equal, with high chances of coinfection in the international border between the territory of Myanmar and the western region of Thailand [G6PD mutation in females follows Hardy–Weinberg equilibrium. Based on the population wide AMM, a total of 17 individuals (6.7%) exhibited intermediate G6PD activity, which was present in 5.7% (14/244) of males and 27.3% (3/11) of females. According to the results of this study and previous report, G6PD deficiency was more common in males than in females whereas intermediate was more common in females than in males [The overall frequency of patients with Thailand , 23. OveThailand , 25. AltThailand , as a reG6PD MahidolG487A mutation was more common among individuals from Myanmar, in Thai, and in Karen malaria patients, whereas G6PD ViangchanG871A mutation was more common among Thai and Cambodian malaria patients. In general, G6PDViangchanG871A is more common in Thai people than G6PD MahidolG487A [G6PD ViangchanG871A and G6PD MahidolG487A in this Thai population. The G6PD MahidolG487A mutation in malaria patients had an MAF of 7.1% in the study population (18 of 255), 7.4% in individuals from Myanmar (14 of 189), 5.7% in Thais (2 of 35) and 12.5% in Karen (2 of 16). These findings agreed with the spatial distribution of G6PD deficient mutations in the Southeast Asia, where G6PD MahidolG487A and G6PD ViangchanG871A mutations are commonly observed on the western and eastern Indochina Peninsula, respectively [G6PD genotype is a key factor of enzyme activity [G6PD coding regions. This could be explained by the methylation of CpG or CpNpG islands on G6PD promotor, resulting in gene silencing [These results showed that the dolG487A . Geneticn 18 of 25, 7.4% iectively , 26, 28.activity , some G6ilencing , 31. Anoilencing .The frequency of PK in this Southeast Asian population was comparable to what was reported by van Bruggen et al., who found this mutation in 13 out of 340 healthy unrelated Southeast Asian subjects with an MAF of 3.2% in individuals from Myanmar, 1% in Thais, 1.5% in Cambodians, 1.8% in Laotians, and 2.9% in Mons . PossiblG6PD MahidolG487A mutation, displayed signs of haemolytic anaemia, including low haemoglobin, RBC count, haematocrit, and high reticulocyte count. However, this study showed no signs of haemolytic anaemia in other G6PD and PKLRR41Q mutations. This is possibly due to the small number of patients enrolled, which limit the chance to detect haemolytic anaemia in malaria patients carrying G6PD AuresT143C, G6PD ViangchanG871A and G6PD KaipingG1388A. According to the World Health Organization (WHO), G6PD variants are categorized based on the degree of enzyme deficiency and severity of haemolysis. G6PD MahidolG487A and G6PD AuresT143C are in a class III mutation (moderately deficient) and G6PD ViangchanG871A and G6PD KaipingG1388A are in a class II mutation (severely deficient) [Correlations between altered G6PD activity due to mutations in malaria patients and haematological phenotypes prior to treatment with anti-malarial drugs have not been well studied. Based on the International Classification of Diseases, 11th Revision (ICD-11) considering classification of G6PD deficiency under haemolytic anaemias code: 3A10.00) , the datA10.00 , ficient) –39.G6PD MahidolG487A was an independent risk factor for anaemia based on age, gender, parasite species, parasite density, PKLRR41Q, thalassaemia, and haemoglobinopathies. G6PD-deficient RBCs are exposed to oxidative stress caused by active neutrophil-produced ROS [in-silico study by Bharti et al., G6PD enzymes with the Mahidol487A mutation lose their crucial catalytic interaction with substrate [G6PD MahidolG487A causes a local conformational change and affects backbone folding. This results in a reduction in thermostability in the absence or presence of NADP+ and a reduction in Kcat, thereby reducing catalytic efficiency [G6PD Mahidol487A mutation, oxidation of membrane proteins causes the cells to rigid, nondeformable, and finally haemolysis. A recent report has indicated that patients with G6PD MahidolG487A presented symptoms of acute haemolytic anaemia after taking an incorrect dose of PQ [G6PD MahidolG487A and other mutations, these findings provided evidence for malaria infection-induced haemolysis in patients with G6PD deficiency. This may be useful for G6PD deficiency testing requirements and administration of anti-malarial drugs including PQ and TQ to prevent relapse of P. vivax and sterilize mature P. falciparum gametocytes with a low risk of adverse events. For radical cure of P. vivax in the Southeast Asia, a high daily dose of PQ (0.5 mg/kg/day) for 14 days has been recommended by the WHO [P. falciparum, a single dose of PQ (0.25 mg/kg) along with artemisinin-based combination treatment (ACT) without a requirement for G6PD deficiency testing has been recommended by the WHO [uced ROS , leadingubstrate . In addificiency , 43. These of PQ . Althoug the WHO . In Thai the WHO . However the WHO . For a g the WHO –48.G6PD MahidolG487A and PKLRR41Q was found in malaria patients in the Southeast Asia with MAFs of 7.1% and 2.5%, respectively. A deficiency in the enzyme G6PD by the G6PD MahidolG487A mutation exhibits a statistically significant correlation with haemolytic anaemia during malaria infection. Together, this study underlines the impact of host genetic background on haemolytic reactions and the benefit of screening for red cell enzymopathies and related mutations in patients before anti-malarial drug administration.In summary, the presence of Additional file 1: Table S1. Mean and standard deviation (SD) for clinical parameters of malaria patients without thalassaemia and haemoglobinopathies


The H9N2 virus can infect not only birds but also humans. The pathogenicity of H9N2 virus infection is determined by an excessive immune response in the lung. All-trans retinoic acid (ATRA), the active metabolite of vitamin A, plays an important regulatory role and has been widely used in the clinical practice. This study was aimed to investigate whether ATRA could regulate the immune response to H9N2 virus infection in the lungs of mice, thereby reducing the pathogenicity of the H9N2 virus in mice.Mice were infected intranasally with H9N2 virus, and injected intraperitoneally with 0.2 mL of ATRA at low (1 mg/kg), medium (5 or 10 mg/kg), or high therapeutic dose (20 mg/kg), and toxic dose , once per day for 10 days. Clinical signs, survival rates, and lung gross pathology were compared between the ATRA-treated H9N2-infected group, the ATRA group, and the H9N2-infected group, to investigate the effect of different doses of ATRA on the pathogenicity of H9N2 virus. Additionally, the viral load and cytokine concentration of lungs were measured at 3, 5, 7, and 9 days after infection, to investigate the potential mechanism of ATRA in affecting the pathogenicity of the H9N2 virus. Expression levels of cellular retinoic acid-binding protein 1 (CRABP1), cellular retinoic acid-binding protein 2 (CRABP2), and Retinoic acid-inducible gene-I (RIG-I) were detected using Western blotting.The ATRA-treated H9N2-infected mice showed more severe clinical signs compared with the H9N2-infected group. The medium and high therapeutic doses of ATRA reduced the survival rates, aggravated lung tissue damage, decreased the expression of interferon beta (IFN-β), and increased the concentrations of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and C-C motif chemokine ligand 2 (CCL2) in the lungs of the H9N2-infected mice. At the same time, the expression patterns of CRABP1, CRABP2, and RIG-I were changed in mice infected by H9N2 and treated with different concentrations of ATRA.Our findings suggest that the therapeutic dose of ATRA can increase the pathogenicity of the H9N2 virus. Therefore, the consequences of those infected by influenza virus would be more severe after ATRA treatment.The online version contains supplementary material available at 10.1186/s12985-022-01809-y. The H9N2 influenza virus, the most widespread subtype of influenza viruses in poultry, has been prevalent in China since 1994, and has transmitted widely around the world in multiple avian species –8, indicAll-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A, plays an important regulatory role in embryonic development, organ growth, metabolism, cell proliferation and differentiation, apoptosis, and immune response regulation –15. ATRAATRA plays its regulatory role via binding to and activating nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) , which aThe aim of our study was to investigate the effects of different doses of ATRA on the pathogenicity of the H9N2 virus in mice, providing guidance for ATRA treatment on the prevention of influenza virus infection, which has important public health implications.To investigate whether ATRA affects the pathogenicity of the H9N2 virus in mice, two sets of experiments were done  in 100 µL per mouse] to simulate a natural infection of H9N2 virus, followed by intraperitoneally injection with ATRA at a low (1 mg/kg), medium (5 or 10 mg/kg), or high therapeutic dose (20 mg/kg), or a toxic dose  at 0–9 days after inoculation (n = 18–22 per group). The mice were monitored for 19 days, and their body weight, food intake, survival rate, and lung gross pathology were compared between the groups.To determine the effect of different doses of ATRA on the pathogenicity of the H9N2 virus, mice were inoculated intranasally with low-dose H9N2 virus [105.5 TCID50 in 100 µL per mouse), followed by intraperitoneally injection of the low (1 mg/kg) or medium (5 or 10 mg/kg) therapeutic dose of ATRA at 0–9 days after inoculation. The mice (n = 22–24 per group) were monitored for 19 days, and their clinical signs , survival rate, pulmonary pathological changes  were compared between the ATRA-treated H9N2-infected group, the ATRA group, and the H9N2-infected group. In addition, lung viral load and cytokine concentrations were measured at 3, 5, 7, and 9 days after H9N2 virus infection (n = 5 per group), to assess the ability of the lungs to clear the virus and the innate immunity level, so as to investigate the potential mechanism of ATRA in affecting the pathogenicity of the H9N2 virus in mice. In all experiments, the mice were treated in the same order each time.To further differentiate the effects of low and medium therapeutic doses of ATRA on the pathogenicity of the H9N2 virus, mice were inoculated intranasally with medium-dose H9N2 virus (10Specific pathogen-free (SPF) male BALB/c mice (6- to 8-week-old) were purchased from the Beijing Vital River Laboratory Animal Technology Company Limited (China). Mice were housed in microisolator cages ventilated under negative pressure with HEPA-filtered air. Water and food were offered ad libitum. All animal experiments were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of the China Agricultural University.2 [50) of ATRA in Swiss mice was 31 mg/kg by intraperitoneal injection [World Health Organization (WHO) recommended oral supplementation at 30–60 mg of ATRA for infant children 6–59 months of age . The dos2 . As reponjection , and the50 on Madin-Darby canine kidney (MDCK) cells, and calculated by the method of Reed and Muench, as described previously [The influenza A virus strain used in this study was A/Chicken/Hebei/4/2008 (H9N2) isolated by our laboratory , 36. Theeviously , 38.5 TCID50 was used as the low-dose H9N2 virus, which would not cause mouse death but significant clinical symptoms, and 105.5 TCID50 was used as the medium-dose H9N2 virus, which could cause death in half of the mice (that is LD50).A low dose of H9N2 virus, which was close to that in natural infection, was adopted to investigate the effect of different doses of ATRA on the pathogenicity of H9N2 virus, observe the interaction between different doses of ATRA and the H9N2 virus, and simulate the infection under natural conditions. In addition, to further differentiate the effects of different therapeutic doses of ATRA on the pathogenicity of the H9N2 virus, a medium dose of H9N2 virus was used. For infection experiments, the H9N2 virus was diluted into different concentrations with sterile PBS, and inoculated intranasally in a volume of 100 µL into mice that were lightly anesthetized with isoflurane. In this study, 1050/mL of lung homogenate. Finally, lung viral load was measured at 3, 5, 7, and 9 days after H9N2 virus infection.Mice were euthanized by isoflurane overdose, and then the whole lungs were removed, weighed, and homogenized in 1.3 mL sterile PBS. The homogenates were centrifuged, filtered through a 0.22 µm cellulose acetate membrane, serial tenfold dilutions made, and then titrated on MDCK cells. Viral loads were reported as the TCIDThe lung homogenates collected at 3, 5, 7, and 9 days after H9N2 virus infection were centrifuged at 4 °C and 5000 rpm for 15 min, and the supernatants were stored at − 80 °C. Concentrations of TNF-α, IL-1β, interferon alpha (IFN-α), IFN-β, CCL2 and C-X-C motif chemokine ligand 10 (CXCL10) were measured using mouse Quantikine ELISA Kits .g, and the supernatants were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked with 3% bovine albumin and probed with monoclonals antibody to β-actin , CRABP1 , CRABP2  and RIG-I , followed by incubation with peroxidase conjugated secondary antibody . Proteins were visualized using enhanced chemiluminescence reagent . The relative protein levels of CRABP1, CRABP2 and RIG-I to β-actin were performed by Image J software.The expression of CRABP1, CRABP2 and RIG-I at protein level was detected by western blotting. As described above, the lung homogenates collected at 3, 5, 7 and 9 days after H9N2 virus infection were lysed with RIPA lysis buffer , quantity of total proteins were measured by BCA kit. The lysates were centrifuged at 14,000Mice were euthanized by overdose of isoflurane 5 days after infection. The lungs were removed, fixed in 10% neutral buffered formalin, and embedded in paraffin. The 5-µm tissue sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H & E) .P values < 0.05 were considered statistically significant.Statistical analyses were performed with the IBM SPSS Statistics v20, and graphics were performed by means of GraphPad Prism software v8.0. Data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparions and the log-rank test for survival rate were conducted. P < 0.05), decreased to the lowest on day 7 , and then quickly returned to normal thereafter. No mice died. Taken together, mice infected with the H9N2 virus showed mild and transient clinical symptoms.The effects of the H9N2 virus on clinical signs and survival in mice were shown in Fig. P > 0.05). The ATRA-treated group (20 mg/kg) had ruffled fur on day 4 after the injection, with reduced activity, loss of appetite, and gradually wasting away. Their body weight and food intake were significantly reduced compared with the blank group (P < 0.05). On day 7, the food intake dropped to the minimum level (decreased by 35.6%). No mice died. The ATRA-treated groups  presented severe clinical signs, including dry skin, rapid wasting, decreased activity, and loss of appetite from 2–3 days after injection. Their body weight and food intake continued to decrease until death. Their body weight and survival were significantly different from those of the blank group (P < 0.01). Taken together, mice showed obvious clinical symptoms when injected with high therapeutic dose of ATRA (20 mg/kg), and died when injected with a toxic dose of ATRA (more than 40 mg/kg).The effects of different doses of ATRA on mouse clinical signs and survival were shown in Fig. P > 0.05, Table P < 0.01) and days 7–8 (P < 0.05), with the lowest on day 5 (decreased by 42.8%) after infection (Table P < 0.05), and decreased to the lowest on day 5 (decreased by 6.9%) and day 4 (decreased by 7.8%), respectively (Table P < 0.05), by 12.2–15.1% and 34.4–60.6% on day 5 after infection, respectively ; however, there was no significant difference in survival rate , 2.0% (P > 0.05), and 2.0% (P > 0.05), respectively; the food intake decreased by 33.4% (P < 0.01), 30.1% (P < 0.01), and 18.1% (P < 0.05), respectively, on day 3 after infection, and the survival rates were significantly different (P < 0.01), except between the 80 mg/kg ATRA group and the 80 mg/kg ATRA-treated H9N2-infected group , and the survival rate was 50%, which was significant different from the blank group  and medium (5 and 10 mg/kg) therapeutic doses of ATRA on the clinical signs and survival rate of H9N2 virus, mice were inoculated with 10P < 0.01). Compared with the 1 mg/kg ATRA group, their body weight was significantly different at 2–10 days and 16–19 days after H9N2 infection , but a significant difference with the 1 mg/kg ATRA group . Compared with the 5, and 10 mg/kg ATRA groups, the body weight of the ATRA-treated H9N2-infected groups also significantly decreased . Compared with the 1, 5, and 10 mg/kg ATRA-treated groups, the lung viral loads of the 1, 5, and 10 mg/kg ATRA-treated H9N2-infected groups significantly increased 3 days after H9N2 infection      , but decreased at 7 and 9 days (P < 0.05)              Fig. a–c, and 05) Fig. d, wherea05) Fig. e, f. Aft05) Fig. e, withou05) Fig. a, and th05) Fig. e. After 05) Fig. a, d, the05) Fig. c, the le05) Fig. e, and th05) Fig. f. Compar05) Fig. f, and th05) Fig. a–e. In t05) Fig. a, the le05) Fig. c, and IF05) Fig. f. In the05) Fig. a, the le05) Fig. c, and th05) Fig. f.Fig. 8EThus, the medium and high therapeutic dose of ATRA could significantly improve the pulmonary inflammatory response of the H9N2-infected mice.As shown in Fig. 50 was 31 mg/kg in Swiss mice after intraperitoneal injection of ATRA for 21 days [In the present study, we investigated whether ATRA could reduce the pathogenicity of the H9N2 virus, by modulating the pulmonary immune response. In the first part, mice were infected with low-dose H9N2 virus to simulate a natural infection of H9N2 virus, followed by the administration of different doses of ATRA. Firstly, we evaluated the clinical manifestations of mice infected with H9N2 virus alone or mice given ATRA alone. We found that compared with the blank group, the body weight and food intake of the H9N2-infected group were significantly reduced on days 5–7, and then quickly returned to normal. Compared with the blank group, the 1, 5, and 10 mg/kg ATRA-treated groups showed no obvious clinical symptoms, the 20 mg/kg ATRA-treated group showed significant body weight and food intake declines, and the 40, 60, and 80 mg/kg ATRA-treated groups showed severe poisoning symptoms, eventually resulting in the death, which were consistent with the results of a previous study by Hixson EJ et al. reporting that the LD 21 days . SecondlIn the second part, we further investigated the effect of the low (1 mg/kg) and medium  therapeutic doses of ATRA on the pathogenicity of increasing doses of H9N2 virus. We found that the low and medium therapeutic doses of ATRA could aggravate the clinical signs and damages to lung tissues in H9N2-infected mice; furthermore, the medium therapeutic dose of ATRA could reduce their survival rate. These results are consistent with the results of our first experiment. Influenza virus infection is characterized by the recruitment of large numbers of innate immune cells to the lungs that release large numbers of proinflammatory cytokines and chemokines . To inveCRABP1, CRABP2, and RIG-I have been reported to be all involved in the process of ATRA regulation of the immune response in mammals. ATRA up-regulates the expression of RIG-I in a RARα-dependent manner, and enhances the signaling cascade mediated by RIG-I, which induces expression of type I IFN and proinflammatory cytokines , 53. CRAAt present, the research on ATRA antiviral effect mainly focuses on (-) ssRNA virus, including measles virus, mumps virus, canine distemper virus and Newcastle disease virus , 56. TheTaken together, our findings suggested an inconsistent result with previous studies on the role of ATRA in pulmonary inflammation. The increased pathogenicity of the H9N2 virus caused by ATRA may be attributed to H9N2 infection and the further treatment of ATRA, which affected the expression patterns of CRABP1, CRABP2, and RIG-I, and in turn resulted in an aggravated immunopathological damage to the lung.The therapeutic dose of ATRA have a potential in increasing the pathogenicity of the H9N2 virus in mice, which might be the underlying reason for the more severe consequences of ATRA treatment for those infected with the influenza virus.Additional file 1: Flow chart of the experiment.


Unfortunately, its on-board application is hampered by the sluggish kinetics, volatile byproducts and harsh conditions for reversibility. In this work, AB and AlH3 were simultaneously infiltrated into a carbon nanotube array (CMK-5) to combine the synergistic effect of alane with nanoconfinement for improving the dehydrogenation properties of AB. Results showed that the transformation from AB to DADB started at room temperature, which promoted AB to release 9.4 wt% H2 within 10 min at a low temperature of 95 °C. Moreover, the entire suppression of all harmful byproducts was observed.Ammonia borane  is considered as one of the most promising hydrogen storage materials for proton exchange membrane fuel cells due to its high theoretical hydrogen capacity under moderate temperatures.Ammonia borane (AB, NH To overcome these obstacles, several strategies have been explored, including the infiltration of AB in porous structures (nanoconfinement),10–12 the addition of metal based catalysts,13–15 modifying thermodynamics through chemical alteration,16–18etc. Among them, nanoconfinement is an effective approach to positively affect the dehydrogenation thermodynamics and kinetics of AB, which originates from the effects of increased surface energy, induced defects and vacancies as well as shortened diffusion distances.19 The pioneering work by Gutowska et al. demonstrated that the decrease of desorption temperature and the suppression of borazine were simultaneously achieved by the capsulation of AB into mesoporous silica.10 However, they didn't mention the problem of NH3 liberation during the decomposition process, which would severely poison PEMFCs even at a very low level (1 ppm).20 This issue could be solved by the combination of nanostructure confinement with metal catalysts. Li et al. incorporated AB into a lithium (Li) functionalized ordered mesoporous carbon framework (CMK-3), and considerable enhancement of dehydrogenation properties as well as complete suppression of all volatile byproducts could be observed in the AB/Li–CMK-3 composite.21 Since then, various surface functionalized nanoscaffolds have been explored, including carbon nanotubes,22–24 metal–organic frameworks,25–27 mesoporous 3D boron nitride,28 Pd/halloysite nanotubes,29etc. On the other hand, the destabilization of AB and suppression of volatile byproducts could also be achieved by compositing with metal hydrides (MH).30–33 Kang et al. found that the mechanically milled mixture of AB and MgH2 could release 8 wt% H2 within 4 h at around 100 °C without the byproducts of diborane and borazine.31 In AB-MH systems, Nakagawa et al. demonstrated that Pauling electronegativity of M is good to indicate the amount of undesired byproducts (NH3 and B2H6), and the level of NH3 decreased with the increase of electronegativity.32 Therefore, AlH3 is more effective than MgH2 on inhibiting the emission of NH3 due to the higher electronegativity of Al. Compared with nanoconfined systems, the dehydrogenation temperatures of these MH-doped composites are relatively high (>100 °C).Owing to the extreme high desorption temperature of step 2 g−1) and bimodal porosity34 , is a p2.38 Alane was synthesized by the organometallic method.40,41 Ammonia borane (97 wt%) purchased from Sigma-Aldrich was used directly without further purification. Before use, CMK-5 was degassed for 20 h at 160 °C to remove adsorbed moisture and gases. Composites of x wt% AB  and CMK-5 with different percentages of AB in CMK-5 were denoted as xAB@CMK-5, and composites of 30 wt% AB and x wt% AlH3  in CMK-5 were denoted as (30AB : xAlH3)@CMK-5. Schematic illustration of the preparation process of (30AB : xAlH3)@CMK-5 is shown in 3 was physically mixed in a mortar by hand-milling and then dissolved in anhydrous THF at room temperature. Afterwards, the THF solution of AB and AlH3 was instilled into CMK-5, and the resulted suspension was further stirred for 2 h at room temperature. Finally, the solvent was evaporated under vacuum overnight. A physical mixture of same amount of AB and AlH3 (denoted as AB/AlH3) was also prepared for comparison. All preparation procedures were carried out under purified argon atmosphere in a glove box or using standard Schlenk technique.CMK-5 was prepared according to the literature.θ–θ diffractometer in Bragg–Brentano geometry with Cu/Kα radiation. The nitrogen adsorption–desorption curves were measured on a NOVA 4200e instrument at −196 °C. All these samples were degassed under vacuum for 24 h at room temperature before measurements. The BET surface areas were obtained from the data in the relative pressure range of 0.05 and 0.20. The pore volumes and pore size distributions were calculated from the desorption branches of isotherms by employing the Barrett–Joyner–Halenda (BJH) model. High-resolution scanning electron microscope (HRSEM) images, and transmission electron microscope (TEM) images were operated on a Hitachi S-5500 ultrahigh-resolution cold field emission scanning microscope. Thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC) measurements were performed on a TGA/DSC STARe system with a heating rate of 5 °C min−1 under Ar atmosphere. The released gas was analyzed using mass spectrometry  with an argon purge rate of 50 mL min−1. IR-spectra were measured with a Fourier transform infrared spectroscopy . Solid-state 27Al nuclear magnetic resonance (NMR) spectroscopy was recorded on a Bruker Avance 500WB spectrometer, using a Doty CP-MAS probe with no probe background. X-ray photoelectron spectroscopy (XPS) measurements were conducted on a Kratos HSI spectrometer with a hemispherical analyzer. The monochromatized Al Kα X-ray source (E = 1486.6 eV) was conducted at 15 kV and 15 mA. An analyzer pass energy of 40 eV was adopted for the narrow scans. The hybrid mode was employed as lens mode. The base pressure in analysis chamber was 4 × 10−7 Pa during the process. The values of binding energy were referred to the C 1s peak (284.5 eV) with the purpose of accounting for charging effects.Wide angle X-ray diffraction (XRD) patterns were recorded on a STOE STADI P diffractometer in Debye–Scherrer transmission geometry with Cu/Kα radiation, and all samples were sealed into glass capillaries with a diameter of 0.5 mm for measurements. Small angle XRD patterns were collected on a Stoe 3.2 adsorption–desorption isotherm, SEM and TEM, and the results are presented in 35 N2 adsorption–desorption analysis @CMK-5. Compared with the obvious diffraction peaks of AB and AlH3 in the AB/AlH3 composite, these peaks become hardly observable after encapsulating into CMK-5 frameworks, indicating the dispersion of AB and AlH3 into mesoporous CMK-5 at a nano or amorphous state. As shown in 3 in CMK-5. 2 adsorption–desorption isotherms and pore size distributions of CMK-5, 30AB@CMK-5 and (30AB : 30AlH3)@CMK-5, respectively. Compared with pure CMK-5, the specific surface areas and pore volumes are declined dramatically from 1650 to 501 and 328 m2 g−1, and from 1.69 to 0.53 and 0.28 m3 g−1, respectively, for 30AB@CMK-5 and (30AB : 30AlH3)@CMK-5 (3.AB and/or AlH3)@CMK-5 . The cor3)@CMK-5 , further3, these confined samples are also characterized by SEM, and the results are shown in 3)@CMK-5 (3. Further energy dispersive X-ray spectroscopy (EDS) mapping results of the surface (3)@CMK-5 indicate the well distribution of AlH3 both in and outside the channels of CMK-5. However, we failed to track the distribution of B and N through EDS due to their light nature and relatively low loaded amount here. In fact, AB was supposed to be well distributed in the inner and external surfaces of CMK-5 through the impregnation method.21,23,40 To further observe the distributions of AB and AlH3 in (30AB : 30AlH3)@CMK-5, TEM was also conducted and the results are presented in 3)@CMK-5 generally displays a rather homogeneous structure. An enlargement of the dotted circle a1 shows that there are some aggregated particles outside the pore channels @CMK-5 is shown in 3 uniformly distribute both in and outside the channels of CMK-5, while a small part of AlH3 gather to form large particles on the surface.To further assess the distribution of AB and/or AlH3)@CMK-5 , no obvi surface  and cros surface  of @CMK-5  are detected in accompany with the liberation of hydrogen above 100 °C. The confinement of AB into CMK-5 contributes to considerable enhancements in the dehydrogenation behavior with an onset dehydrogenation temperature as low as 50 °C and two decomposition peaks at 98 °C and 140 °C. Moreover, a significant decrease of ammonia and a complete suppression of diborane and borazine are observed. These improvements are correlated with the nanoconfinement effect of CMK-5, which affects the decomposition thermodynamics and kinetics of AB.41 Notable reductions of dehydrogenation temperature and byproducts are also observable in AB/AlH3. However, the liberation of NH3 is still detectable during the heating process of 30AB@CMK-5 and AB/AlH3. Further inhibition of NH3 can be realized through the synergistic effect of CMK-5 and AlH3. As shown in Fig. S1,3 decreases with increasing the amount of AlH3 in (30AB : xAlH3)@CMK-5 . A complete suppression of NH3 can be achieved by loading the same amount (30 wt%) of AB and AlH3 into CMK-5. As shown in 3 and (30AB : 30AlH3)@CMK-5 are obviously lower than neat AB, further indicating the suppression of byproducts. Because of partly liberated NH3, the weight loss from 30AB@CMK-5 is much higher than that of (30AB : 30AlH3)@CMK-5 under the same conditions. 3 and (30AB : 30AlH3)@CMK-5. This indicates that the first equivalent H2 is released prior to the melting of AB in these samples.42 On the basis of integral calculation from DSC curves, the decomposition reaction enthalpies of neat AB and AB/AlH3 were calculated to be −19.9 and −19.7 kJ mol−1, respectively, which is in agreement with reported values (−21 kJ mol−1) for pure AB.10,21,43 The decomposition reaction enthalpies of AB in 30AB@CMK-5 and (30AB : 30AlH3)@CMK-5 increase to −2.9 and −2.0 kJ mol−1, respectively, due to the destabilization effect of nanoconfinement. 3)@CMK-5 at different temperatures. For comparison, the dehydrogenation curves of neat AB at 85 °C and 30AB@CMK-5 at 65 °C were also presented. Even at low temperatures of 65 °C and 55 °C, AB in (30AB : 30AlH3)@CMK-5 can liberate 2.3 wt% and 1.2 wt% H2 within 10 min. Increasing temperature to 85 °C, a capacity of 6.8 wt% H2 can be released in 10 min while only a very small amount of hydrogen (0.2 wt%) is detected from neat AB within the same time. At temperature up to 95 °C, the amount of released hydrogen in 10 min increases to 9.4 wt% and reaches 10.5 wt% in 30 min. These results indicate a kinetic improvement of the decomposition process of AB through the synergistic effect of nanoconfinement and alane.MS, TGA, DSC and isothermal measurements were conducted to compare the dehydrogenation behaviors of AB, 30AB@CMK-5, AB/AlH3)@CMK-5 . All the11B NMR and XPS spectra of 30AB@CMK-5 and (30AB : 30AlH3)@CMK-5 before and after heating to 80 °C and 100 °C were recorded, and the results are presented in 2 and BH4 of the diammoniate of diborane (DADB), [(NH3)2BH2+][BH4−]. DADB is a reactive product, the production of which is a crucial nucleation event that can trigger the rapid release of hydrogen, but its formation in pure AB required a heating period of 30 min at 85 °C.44 In our case, the transformation from AB to DADB starts in 30AB@CMK-5 during the impregnation or drying process at room temperature, demonstrating the destabilization effect of nanoconfinement on AB. Usually nanoscaffolds containing reactive oxygen functional groups can react with AB during the heating process.10,21,45 XPS results also show hydroxyl groups in CMK-5  species after the release of one equivalent of H2 from AB. No borazine compound can be observed at 31.1 ppm.47 For (30AB : 30AlH3)@CMK-5, as shown in 4 position from −37.6 ppm to −40.2 ppm, suggesting that the addition of AlH3 polymerizes DADB. 3)@CMK-5 at different temperatures. The peaks at ∼189.5 eV correspond to B–O bond, which is close to a structure with oxidized trigonal geometry ,48 further confirming the reaction between AB and surface hydroxyl groups. After thermal decomposition at 100 °C, the peak associated with B–H bond appears at ∼185.2 eV in 30AB@CMK-5 (3)@CMK-5 (3)@CMK-5. From the XPS elemental composition analysis, as shown in 3. While there is almost no change of the N content in (30AB : 30AlH3)@CMK-5 under the same condition, this demonstrates the immobilization of N in this composite. These results indicate that, on the one hand, AB reacts with surface located functional groups to form organic borates (–OBX), and further addition of AlH3 would change the decomposition route of confined AB in (30AB : 30AlH3)@CMK-5 because of coulombic attraction between the hydridic Hδ− of AlH3 and protonic Hδ+ of NH3 moieties in AB.32,49 On the other hand, the hydridic Hδ− of AlH3 and protonic Hδ+ of NH3 moieties in DADB also results in the polymerization of DADB as –NH2BH2NH2BH4–. The interaction between Al–H and N–H can be further confirmed by the FT-IR spectrometry in Fig. S5.3)@CMK-5. As shown in Fig. S5a,3)@CMK-5, the intensity of N–H bonds also decline whereas the stretching bonds of B–N are almost constant with only a slight shift of the positions @CMK-5 . Besides3)@CMK-5 can be illustrated in via the reaction between –BH3 and –OH to generate –OBH2 species, resulting in the cleavage of B–N and B–H bonds to liberate H2 and NH3; (2) AB molecules inside CMK-5 channels would release H2 through the pathway from DADB to PAB, PIB and H2 because of the nanoconfinement effect of CMK-5. As shown in 3 and NH3 in (30AB : 30AlH3)@CMK-5 could influence these two decomposition steps as follows: (1) coulombic attraction between the hydridic Hδ− of AlH3 and the protonic Hδ+ of NH3 moieties in AB would result in the cleavage of B–H and N–H bonds to prompt the production of –OBNH2– like structures, which is responsible for the immobilization of N and thus the suppression of NH3; (2) hydridic Hδ− of AlH3 reacts with active DADB to polymerize as –NH2BH2NH2BH4– species, leading to the stabilization of BH4 during the heating process.According to the abovementioned analysis, the decomposition pathways of 30AB@CMK-5 and @CMK-5, has been fabricated for the possible applications in combination with low temperature PEMFCs. Results demonstrated that the synergetic effect of nanoconfinement of CMK-5 and AlH3 in (30AB : 30AlH3)@CMK-5 dramatically improve the dehydrogenation thermodynamics and kinetics of AB, meanwhile suppress the liberation of byproducts. AB in this framework composite can release 9.4 wt% H2 within 10 min at a low temperature of 95 °C, and the dehydrogenation enthalpy increases to −2.0 kJ mol−1 from the pristine value of −19.7 kJ mol−1. This advancement demonstrates that the combination of nanoconfinement with MH could be further explored to improve the hydrogen storage properties of AB and/or other hydride systems.In summary, a new AB and AlHThere are no conflicts to declare.RA-010-D0RA02283G-s001


SLC5A7 (solute carrier family 5 member 7), also known as choline transporter 1 (CHT1), is downregulated in colorectal cancer (CRC) and functions as a tumor suppressor. However, the mechanisms underlying the inactivation of SLC5A7 in CRC remain to be elucidated.In the present study, two broad-spectrum demethylation agents (azacitidine and decitabine) employed to treat CRC cells significantly upregulated SLC5A7 expression. Further results based on the CRC cohort and TCGA database indicated that SLC5A7 promoter methylation inversely correlated with SLC5A7 expression, and the status of SLC5A7 promotor methylation showed a promising prognostic value for patients with CRC. Next, the dCas9-multiGCN4/scFv-TET1CD-based precision demethylation system was constructed, which could significantly and specifically promote SLC5A7 expression in CRC cells through sgRNA targeting the SLC5A7 promoter. Both in vitro and in vivo experiments demonstrated that targeted demethylation of SLC5A7 by dCas9-multiGCN4/scFv-TET1CD-sgSLC5A7 inhibited tumor growth by stabilizing p53 and regulating downstream targets.Collectively, DNA promoter methylation caused inactivation of SLC5A7 in CRC, and targeted demethylation of SLC5A7 might be a therapeutic target for CRC and other cancers.The online version contains supplementary material available at 10.1186/s13148-022-01308-5. DNA methylation plays a pivotal role in gene regulation at the transcriptional level, and alterations in methylation regulate diverse pathological processes, including cancer initiation and progression –3. Hence+-dependent choline transporter with 13 transmembrane domains, is mainly expressed in the spinal cord and medulla (intracellular vesicles), and belongs to the Na+/glucose co-transporter family (SLC5) [SLC5A7 is a Nay (SLC5) –21. SLC5y (SLC5) , 23. Muty (SLC5) –27. In cy (SLC5)  revealedy (SLC5) . More imy (SLC5) . HoweverIn this study, we aimed to clarify the mechanism underlying the inactivation of SLC5A7 in CRC and the potential correlation between the methylation of the SLC5A7 promoter and prognosis of individuals with CRC. Importantly, we constructed a system based on dCas9-multiGCN4/scFv-TET1CD-sgRNA that targets DNA demethylation at specific loci to reverse SLC5A7 expression level and verified its efficacy and specificity. Moreover, in vitro and in vivo experiments were employed to determine the potential antitumor effect and mechanism of dCas9-multiGCN4/scFv-TET1CD-sgSLC5A7 in CRC. Our results would provide solid evidence for understanding the mechanisms underlying SLC5A7 downregulation and its role in CRC, and a potential therapeutic strategy for CRC, even for other cancers.2. All cancer cells were authenticated periodically by DNA profiling on a common platform of the Scientific Research Center, West China Hospital, to ensure that they were mycoplasma-free. Azacitidine  and decitabine  were purchased from Selleck  and used for HCT116 and RKO treatment with a final concentration of 10 μM and 1 μM separately. At 24 h post AZA and DAC treatment, the cells were collected for further analysis.Human CRC cells (HCT116 and RKO) were obtained from American Type Culture Collection  and then cultured in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium  supplemented with 1% penicillin–streptomycin  antibiotics and 10% fetal bovine serum  at 37 °C in a moisturized atmosphere containing 95% air and 5% COThe antibodies used in this study were as follows: SLC5A7 , p53 , phospho-p53 , p21 , IGFBP3 , CDK6 , Bax , and GAPDH .6 cells per mouse). Tumor volume (mm3) was calculated using the following formula: volume (mm3) = width (mm) × length2 (mm) × 0.52. Tumors from each mouse were collected after they were killed and weighed.Five to six-week-old female BALB/c nude mice were obtained from Gempharmatech Co., Ltd.  and allowed to adapt in a SPF condition for a week before use. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of West China Hospital, Sichuan University (No. 2021417A), and conformed to IACUC’s regulatory codes. Mice were raised in ventilated cages with a maximal number of 5 mice per cage and provided with adequate water and food. Housing environment was kept at a temperature between 20–24 °C and 30–70% humidified atmosphere. Twelve-hour light–dark cycles were applied to mimic natural day and night shift. To create a subcutaneous cancer model, control sgRNA and sgSLC5A7 HCT116 and RKO cells were subcutaneously implanted into the right flank of nude BALB/c mice  and disease-free survival (DFS) of patients were recorded for more than 5 years. This part of the study was approved by the Ethics Committee of West China Hospital, Sichuan University .2O to meet 10 μL in volume. The reaction mixture was then incubated for 15 min at 50 °C. To select the stably transformed clones, competent Escherichia coli was combined with 5 μL reaction mixture indicated above and disseminated over a solid medium containing kanamycin. Finally, the plasmid was purified, and the colony was cultured. The targeted sequences were as follows:sgSLC5A7-1: CACCGACACCCCTCGCGGTTCCAGG;sgSLC5A7-2: CACCGGATACCATGGCTGGCAGCG;sgSLC5A7-3: CACCGTGCGGGATGCGAAGGAAACG;sgSLC5A7-4: CACCGCCTCGCACCCACACCCCTCG;sgSLC5A7-5: CACCGAAAAGTCCCCTTTATAAGGG;sgSLC5A7-6: CACCGATGCTCTCCCGGCCCCCTG.A vector system (Addgene plasmid 82559), which contains three fundamental components: Cas9 peptide sequence (linker length: 22aa), antibody-sfGFP-TET1CD, and gRNA , was useFor transfection, HCT116 and RKO cancer cells were cultured in 6-well dishes  and X-tremeGENE HP DNA Transfection Reagent  was used following the manufacturer’s protocols for plasmid transfection into cells. G418 (1000 μg/mL) was added to the culture to select successfully transfected cells with a demethylation system. Four days after transfection, the cells were collected to extract genomic DNA and RNA to detect target gene expression and DNA methylation levels. For selecting the stable transfected cells used for animal study, G418 was added to the transfected cells and the concentration was increased from 1000 to 3000 μg/mL, last for 12 days.The siRNAs for DNMT1, DNMT3a, DNMT3b, and siNC were purchased from Riobio, Co., Ltd., Guangzhou, China. All procedures were consistent with the manufacturer’s protocol, and 48–72 h later, cells were obtained to test the transfection efficiency.−ddCt method.Whole RNA was extracted from cancer cells using TRIzol reagent . Reverse transcription of the extracted RNA into cDNA was achieved using the PrimeScript™ II 1st Strand Synthesis Kit . The acquired complementary DNA replicates were further analyzed using SYBR Green Master Mix  following the reaction conditions of 95 °C (300 s), followed by 45 cycles of 95 °C for 5 s, 58 °C for 30 s, 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. Quantitative real-time PCR was performed using the QuantStudio3 PCR system , and relative mRNA abundance was calculated using the 2Cancer cells were collected after transfection with sgRNA or siRNA. Then, using RIPA lysis buffer  containing 1% protease inhibitor cocktail , total protein was extracted on ice for 30 min. Next, the BCA Protein Assay Kit  was used to determine the protein concentration. Further, using SDS-PAGE gel electrophoresis, 10 μg of extracted proteins with loading buffer was separated and transferred to a PVDF membrane. The PVDF membranes were blocked with 5% skim milk, followed by incubation at 4 °C overnight with antibodies against SLC5A7 , p53 , phospho-p53 , p21 , IGFBP3 , CDK6 , Bax , and GAPDH . The next day, secondary antibodies labeled with horseradish peroxidase  were incubated for 1 h, and visualization was performed using an ECL western blotting substrate kit .A Tissues & Cell Genomic DNA Purification Kit  was used to extract genomic DNA from cells and samples, and the modified DNA was used for time-of-flight mass spectrometry, which was performed by Oebiotech Co., Ltd., Shanghai, China.Cell Counting Kit 8  was used to measure cell viability. In brief, approximately 1000 cancer cells per well, with five replicates, were seeded in 96-well plates and incubated. After 72 h of incubation, 10 μL of CCK-8 reagent was added to the culture and incubated for 3–4 h. Then, the absorbance at 450 nm was measured for each well. In the colony-formation assay experiments, approximately 1000 cells per well were seeded in triplicate in six-well plates and incubated for 8–10 days. When visible cell colonies were detected, each well was gently washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde. Finally, after staining with 0.1% crystal violet, the visible colonies were counted under an inverted microscope. Each experiment was repeated at least three times.IHC staining of paraffin-embedded tissues of subcutaneous tumors was performed with primary antibodies against SLC5A7 , p53 , and phospho-p53  in a moist dark chamber at 4 °C for 8–10 h. Then, these specimens were incubated with secondary antibody for approximately 2 h and stained with DAB . Positive cells were deemed to be cells with moderate to strong brown staining in both the nucleus and cytoplasm.t and Chi-square tests were used for further comparisons, and Pearson’s correlation analysis was performed to determine the correlation between SLC5A7 expression and methylation level. Statistical significance was set at p ≤ 0.05. All statistical analyses were performed using the GraphPad Prism 5 .All experiments were conducted 3–5 times to ensure a consistent outcome. Kaplan–Meier survival analysis of disease-free survival (DFS) and overall survival (OS) was performed using the median value as the cutoff point, and significance comparison was examined using the log-rank test. All data are presented as the mean ± SD. Two-tailed unpaired Student’s Our previous study showed that SLC5A7 is downregulated in the majority of CRC tissues and cells . To furtp < 0.001, Fig. p < 0.001, Fig. n = 69, p = 0.0077 and p = 0.0067, respectively) and TCGA-COAD cohort   [For targeting the SLC5A7 promoter region from − 621 to 939 bp, six sgRNAs were designed to construct dCas9-multiGCN4/scFv-TET1CD-sgRNAs, which could specifically demethylate the SLC5A7 gene to reverse its expression    to deter6 HCT116 cells into the right flank of female BALB/c nude mice showed that HCT116 cells stably transfected with sgSLC5A7-2 and sgSLC5A7-3 had significantly smaller tumor volume  at transcription start sites inhibit subsequent gene transcription , 33. IndBecause of some undesirable side effects resulting from the application of global demethylation agents such as AZA and DAC, more precise and effective strategies for demethylation are needed. Thus, the dCas9-multiGCN4/scFv-TET1CD-sgRNA-based system that targets the selected DNA methylated loci was applied. There are also other methods such as ZFs -based toCholine is crucial in the formation and biological function of cell membranes and cholinergic neurotransmission, because it is a fundamental resource of phospholipid phosphatidylcholine and the synthesis of the neurotransmitter acetylcholine (ACh) , 36. SLCIn conclusion, our results demonstrated that the DNA methylation level mediates SLC5A7 expression and could be used as a prognostic indicator for CRC patients. Next, by using dCas9-multiGCN4/scFv-TET1CD-sgSLC5A7 that specifically demethylates the SLC5A7 promoter region, the expression of SLC5A7 could be restored. The implementation of targeted SLC5A7 DNA demethylation reagents resulted in significant anti-tumor effects both in vitro and in vivo. These anti-tumor effects have been demonstrated to be associated with the stabilization of p53 and its downstream regulators. Moreover, the dCas9-multiGCN4/scFv-TET1CD-sgSLC5A7 may have promising therapeutic value in colon cancer and other cancers.Additional file 1: Fig. S1. Specificity of siDNMT1, siDNMT3a, and siDNMT3b. (A) Expression determination of DNMT1 after transfection of siNC, siDNMT1, siDNMT3a, and siDNMT3b in HCT116 cells by qPCR . (B) Expression determination of DNMT3a after transfection of siNC, siDNMT1, siDNMT3a, and siDNMT3b in HCT116 cells by qPCR . (C) Expression determination of DNMT3b after transfection of siNC, siDNMT1, siDNMT3a, and siDNMT3b in HCT116 cells by qPCR .Additional file 2: Fig. S2.. Off-target analysis of the dCas9-based specific demethylation system. (A&B) 8 and 5 potential off-target sites predicted for sgSLC5A7-2 and sgSLC5A7-3, respectively, using a web-based system as preciously described [escribed . (C&D) q