In cells treated with these molecules, we systematically evaluated the expression of BCL-2 family members, autophagy progression, mitochondrial network structure, regulation of mitochondrial fusion/fission, reactive oxygen species, and ATP production. We found that these compounds affect autophagy induction to prevent cardiac cell death and can be promising cardioprotective drugs during chemotherapy.Improvement of anticancer treatments is associated with increased survival of cancer patients at risk of cardiac disease. Therefore, there is an urgent need for new therapeutic molecules capable of preventing acute and long-term cardiotoxicity. Here, using commercial and home-made chemolibraries, we performed a robust phenotypic high-throughput screening in rat cardiomyoblast cell line H9c2, searching for small molecules capable of inhibiting cell death. A screen of 1600 compounds identified six molecules effective in preventing necrosis and apoptosis induced by H One of the major problems in anticancer treatments is the management of toxicity that affects cardiac cells and leads to cardiac dysfunction and cardiomyopathy in many surviving patients. The number of patients at risk for cardiovascular diseases increases in correlation with the improvement of survival for most cancers resulting in higher cardiovascular morbidity and mortality ,2.Acute cardiac damages can be induced by tissue irradiation and chemotherapy, especially upon treatment with tyrosine kinase inhibitors and anthracyclines (doxorubicin and epirubicin), as often observed in childhood cancer survivors ,3,4,5,6.2O2, which causes oxidative damage and induces both necrosis and apoptosis [2O2 or camptothecin and further characterized their cellular and molecular effects in rat primary neonatal cardiomyocytes (RNVCs). To be effective, all molecules require autophagy regulators ATG5 and BECLIN-1 proteins but have differential abilities to regulate cell death, autophagy, and mitochondrial structure. Overall, these compounds are promising cardioprotective drugs to be used in the course of chemotherapy and should be further tested during preclinical studies.Here, in perspective to find novel cardioprotective drug candidates, we performed a phenotypic high-throughput screening using a rat cardiomyoblast cell line, H9c2, and tested commercial and home-made library of 1600 molecules searching for compounds capable of inhibiting both apoptosis and necrosis. We used camptothecin, a potent apoptosis inducer that acts both as the DNA-intercalating agent and topoisomerase I inhibitor  and H2O2Compounds obtained from Prestwick library (1200 molecules) and CEA SCBM library (400 molecules) were dissolved at 10 mM in 100% DMSO to prepare stock solutions. The distribution of compounds into 96 well plates was made with a Biomek Single Bridge 96 liquid handler . 2O2  2 h to induce necrosis, and 0.1% DMSO in culture medium was used as a negative control.H9c2 cells (ATCC 30-2002™) were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with Fetal Bovine Serum 10%  and penicillin-streptomycin mixture . H9c2 cells were seeded in 96 well plates (5.000 cells/well), let adhere for 48 h, and treated with compounds at 10 µM for 2 h at 37 °C. Compounds were removed and replaced with a culture medium containing either 10 µM camptothecin  24 h to induce apoptosis or 300 µM HThe percentage of viable cells was evaluated by methylene blue staining . After t2+, 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2% HEPES (pH 7.6) and plated on culture dishes, coated with 10 μg/mL laminin. RNVCs were left to adhere for 2 h in a 95% O2, 5% CO2 at 37 °C before the medium change.Rat neonatal cardiomyocytes (RNVCs) were isolated as previously described . BrieflyA colorimetric assay was used to measure lactate dehydrogenase (LDH), a cytosolic enzyme released upon plasma membrane permeabilization, and to evaluate cell viability. Assay from Promega was performed using cell culture supernatants obtained from H9c2 cells or RNVCs, and LB was used as a positive control of total cell lysis. LDH release was measured at 490 nm .Propidium iodide , a fluorescent impermeable DNA marker, was used to measure plasma membrane integrity. Propidium iodide at 10 µM was added in the culture medium, and fluorescence reading was performed  using TECAN infinite spectrofluorimeter , commercial lysis buffer (LB) was used as a control.5 neonatal cardiomyocytes were plated overnight on 35 mm culture dishes coated with 10 µg/mL laminin, and 24 h later, cells were transiently transfected with 1 µg plasmid coding for GFP-LC3  by using 2.5 µL Lipofectamine® 2000  for 48 h. Fluorescence was detected with a confocal microscope (SP5 Leica). Images were analyzed with Image J .Then, 4 × 105 RNVCs were plated overnight on 35 mm culture dishes coated with 10 µg/mL laminin, and 24 h later, cells were treated for 6 h with 1 µM or 10 µM of different compounds. Cells were incubated with Mitotracker Red 580 at 200 nM for 20 min at 37 °C, then with 4 μM calcein  for 10 min at 37 °C. Z stack images were acquired with a Leica (TCS SP8 gSTED) inverted confocal laser scanning microscope  equipped with a WLL Laser . Green fluorescence emission was detected with 505–550 nm wide emission slits and 585–700 nm wide emission slits for the red signal under a sequential mode. The pinhole was set at 1.0 Airy unit, and 12-bit numerical images were done with the Leica Application Suite X software .4 × 10Mitochondrial network and cell volume 3D model were reconstructed by using the IMARIS software 9.7 version ; consequently, cell volume, mitochondria number, and volume were analyzed using the volume and surface rendering processes.For ultrastructural analysis, cells were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, washed in 0.1 M cacodylate buffer, fixed for 1 h in 1% osmium tetroxide, and 1% potassium ferrocyanide in 0.1 M cacodylate buffer to enhance the staining of membranes . Cells wA total of 50 μg of MitoSOX mitochondrial superoxide indicator  was dissolved in 13 μL of DMSO to make 5 mM MitoSOX™ stock solution, which was further diluted in PBS to make a 5 μM MitoSOX working solution. RNVCs were treated with either 0.1% DMSO or 3 μM rapamycin, or 1 μM solutions of digitoxigenin, digoxin, SG6163F VP331, LOPA87, or minaprine in cell culture medium for 6 h. After treatments, cells were washed 2 times with PBS at 37 °C, incubated with 5 μM MitoSOX for 10 min at 37 °C, and gently washed three times with warm PBS. The nuclear fluorescence was deleted, and mitochondrial fluorescent intensity was measured by using ImageJ software.2, 2 mM MgSO4, 1.2 mM NaH2PO4, supplemented on the day of the assay with 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES pH 7.4), was kept at 37 °C. H9c2 cells were seeded at 20,000 cells per well in XF96 cell culture microplates; all the pre-treatments were performed with a serum-free cell culture medium. A total of 24 h prior to the assay, the growth medium was replaced with the substrate-limited medium, and 45 min prior to the assay, cells were washed two times with FAO Assay Medium; 150 μL/well FAO Assay Medium was added to the cells and incubated in a non-CO2 incubator for 30–45 min at 37 °C. The assay cartridge was loaded with XF Cell Mito Stress Test compounds . Finally, 30 μL XF Palmitate-BSA FAO Substrate or BSA was added to the appropriate wells, then immediately inserted the XF Cell Culture Microplate into the XFe96 Analyzer for analysis.The XFe96 Extracellular Flux Analyzer  was used to measure cellular bioenergetic function. H9c2 cells were seeded at 20,000 cells per well in XFe96 cell culture microplates; all the pre-treatments were performed with a serum-free cell culture medium. The Agilent Seahorse XF Glycolysis Stress Test Kit  was used to measure glycolytic function by quantification of the extracellular acidification rate (ECAR) followed by 3 sequential injections of 10 mM glucose, 2 µM oligomycin, and 50 mM 2-deoxy-D-glucose. The oxygen consumption rate (OCR) was measured with Seahorse XF Cell Mito Stress Test Kit . The built-in injection ports on XF sensor cartridges were used to add modulators of respiration into cells during the assay to reveal the key parameters of mitochondrial function. Then, 2 µM oligomycin was injected first, followed by the addition of 1 µM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP). Finally, 0.5 µM antimycin A was injected to stop mitochondrial respiration. The oxidation of exogenous fatty acids was measured using the XF Palmitate-BSA FAO Substrate kit  and the XF cell Mito Stress Test kit. Cells were grown in DMEM supplemented with 0.5 mM glucose, 1 mM GlutaMAX, 0.5 mM carnitine, and 1% fetal bovine serum. The FAO Assay Medium , incubated for 5 min at 95 °C, and loaded in 4–20% Tris-Glycine gel . Separated proteins were transferred onto PVDF membrane for 3 min at 2.5 V in Trans Blot Turbo System . The membrane was blocked with 5% milk in PBS/0.1% Tween and incubated overnight with an appropriate primary antibody in 5% milk in PBS/0.1% Tween at 4 °C. The membrane was washed 6 times × 5 min with PBS/0.1% Tween, incubated with a Horseradish Peroxidase-Conjugated secondary antibody for 1 h at room temperature, washed again with PBS/0.1% Tween, incubated with an ultra-sensitive enhanced chemiluminescent substrate for 5 min, and visualized with a gel imaging system . The following antibodies were used: anti-Mitofusin 1 , anti-Mitofusin 2 , BCL-2 (C-2) , BAX (B-9) , BCL-XL , LC3B (D11) , β-actin (C4) , phospho-DRP1 (Ser616) (D9A1) , and DRP1 .H9c2 cells and RNVCs were detached in LB containing 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% deoxycholate, 1% Triton X 100, and 0.1% SDS. The cells were collected, placed on ice for 30 min and centrifuged at 2000× p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Results are expressed as mean ± standard error (SD) or standard error to the mean (SEM). The Origin software and Graphpad Prism 6 were used for statistical analysis. Differences between 2 groups were analyzed by one-way ANOVA, and differences between groups of two genotypes were analyzed by two-way ANOVA, Sidak’s multiple comparisons. Statistical significance is indicated as follows: * 2O2-induced necrosis and camptothecin-induced apoptosis in rat cardiomyoblast H9c2 cell line, a phenotypic high-throughput screening was performed with 1200 molecules from the commercial library Prestwick and 400 molecules from the home-made chemical library  using LDH assay and propidium iodide staining , suggesting that selected compounds can induce autophagy as a cytoprotective mechanism. We next measured the capacity of compounds to activate autophagy following the conversion of cytosolic LC3 I to autophagosome associated LC3 II and found that LC3 II expression level was significantly increased by 1 μM SG6163F (>1.5 fold) and 3 μM rapamycin (>1.4 fold) treatment  and chloroquine (CQ) . In the Altogether, these results reveal that all compounds require ATG5 and BECLIN1 to exert their cell death inhibitory activity, but only SG6163F stimulates the autophagic flux. We next verified the effects on mitochondria in cells treated with different compounds because these organelles play a major role in cardiomyocyte cell functioning ,19,20. FIn digoxin and SG6163F- treated cells, numerous short and round mitochondria can be observed compared to 0.1% DMSO-treated cells (controls), which have long and thin mitochondria . Thus, tTo dissect the metabolic effects of compounds, we analyzed the energy metabolism of H9c2 cells in real time. We found that all compounds except rapamycin increased extracellular acidification suggesting an increase of ATP productions by anaerobic glycolysis A. DigitoFinally, we evaluated ROS production by detecting anion superoxide in RVNCs by MitoSOX fluorescent probe following cell treatment by the compounds during 6 h. We found that rapamycin, digitoxigenin, VP331, LOPA87, and minaprine but not digoxin or SG6163F induced a local mitochondrial anion superoxide production in line with the observed activation of OXPHOS .By high-throughput screening, we have identified new compounds capable of inhibiting cardiac apoptosis and necrosis and characterized their effects in H9c2 cells and in primary RNVCs. Among these compounds, digitoxigenin and digoxin (cardiac glycosides) and minaprine are molecules from Prestwick library, a commercial library of 1200 off-patent small molecules, 95% being approved drugs. Three other chemicals, SG6163F, VP331, and LOPA87, are new small molecules, which were synthesized in our laboratories ,14.+,K+-ATPase. Cardiac glycosides have increased sensitivity in cancer cells [Cardiac glycosides are natural molecules used in clinical medicine known for their antagonistic action on Naer cells  and haveer cells , promoteer cells , and meder cells . Here, wer cells , our reser cells .2O2-induced oxidative alterations and DNA-damage events induced by camptothecin. Indeed, all compounds required BECLIN-1 and ATG5 to protect primary cardiomyocytes from cell death, as shown by the downregulation of these autophagy activators upon siRNA treatment , which might hamper preclinical studies in animals . Therefo


HT22 cells were established as a model of oxygen–glucose deprivation and reoxygenation (OGD/R) with high glucose to mimic hyperglycemia and I/R in vitro. HT22 cells were treated with/without different concentrations of the UCP2-specific inhibitor genipin for different periods of time. The results showed that UCP2 deficiency significantly increased histopathological changes and apoptosis after cerebral I/R damage in hyperglycemic mice. Moreover, UCP2 deficiency enhanced NLRP3 inflammasome activation in neurons when cerebral I/R damage was exacerbated by hyperglycemia. Furthermore, UCP2 deficiency enhanced NLRP3 inflammasome activation and reactive oxygen species (ROS) production in HT22 cells under OGD/R and high-glucose conditions. UCP2 deficiency aggravated hyperglycemia-induced exacerbation of cerebral I/R damage. UCP2 deficiency also enhanced NLRP3 inflammasome activation and ROS production in neurons in vitro and in vivo. These findings suggest that UCP2 deficiency enhances NLRP3 inflammasome activation following hyperglycemia-induced exacerbation of cerebral I/R damage in vitro and in vivo. UCP2 may be a potential therapeutic target for hyperglycemia-induced exacerbation of cerebral I/R damage.Mitochondrial uncoupling protein 2 (UCP2) deficiency exacerbates brain damage following cerebral ischemia/reperfusion (I/R). The Nod-like receptor protein-3 (NLRP3) inflammasome also plays a vital role in cerebral I/R damage. However, the effect of UCP2 on NLRP3 inflammasome-mediated hyperglycemia and I/R damage is not clear. In the present study, UCP2-knockout (UCP2 Ischemic stroke is identified as one of the major causes of disability and mortality worldwide. Hyperglycemia is an important independent risk factor for ischemic stroke and induces the occurrence and development of cerebral I/R damage , 2. PrevUCP2 is the inner membrane protein of mitochondria. It has been proposed that UCP2 regulates the mitochondrial potential and reactive oxygen species (ROS) production . To dateThe inflammasome plays an important role in neuroinflammation. The NLRP3 inflammasome is a multiprotein complex that comprises NLRP3, ASC and caspase 1. NLRP3 inflammasome activation leads to NLRP3 recruitment of ASC and pro-caspase 1, which causes caspase-1 activation and the release of caspase activation-dependent cytokines, including IL-1β and IL-18. NLRP3 inflammasome activation may trigger or exacerbate neuroinflammation, thereby promoting cerebral ischemia injury , 8.−/−) and wild-type (WT) mice to establish a model of middle cerebral artery occlusion (MCAO) and reperfusion under normo- and hyperglycemic conditions in vivo. HT22 cells were established as a model of oxygen–glucose deprivation and reoxygenation (OGD/R) with high glucose to mimic hyperglycemia and I/R in vitro. HT22 cells were treated with/without different concentrations of the UCP2-specific inhibitor genipin for different times. We evaluated the effects of UCP2 deficiency and the activation of the NLRP3 inflammasome in hyperglycemia-induced exacerbation of cerebral ischemic injury.In this study, we hypothesized that UCP2 deficiency enhances NLRP3 inflammasome activation after hyperglycemia-exacerbated cerebral I/R damage. Thus, we used UCP2-knockout (UCP2A TUNEL assay kit (#11684817910) was purchased from Roche . Streptozotocin (STZ) was purchased from Sigma. An ROS assay kit (#S0033) was obtained from Beyotime . NLRP3 (ab214185), NeuN (ab104224) and IL-1β (ab9722) antibodies were purchased from Abcam . ASC (D2W8U) and TXNIP (D5F3E) antibodies were obtained from Cell Signaling Technology . Caspase 1 (AF5418) and cleaved caspase-1 (AF4005) antibodies were purchased from Affinity.β-Actin and GAPDH antibodies were purchased from Bioss . Genipin  was diluted to appropriate concentrations in cell culture medium. ELISA kits were purchased from Abcam.−/− mice on a C57BL/6 background were originally obtained from the Jackson Laboratory (USA). UCP2−/− mice were fed and bred in sterile animal housing, and age-matched male mice were used for the experiments. Male wild-type (WT) C57BL/6 mice (6–8 weeks old) were obtained from Vital River Laboratory . All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of Ningxia Medical University and the ethical review committee of Ningxia Medical University . Animal experimental procedures were performed according to the NIH Guide for Care and Use of Laboratory Animals.Breeding pairs of UCP2−/− mice were used. UCP2−/− and WT mice were randomly divided into the sham (sham-operated) group, NG + I/R (normoglycemic + 1 h MCAO and 24 h reperfusion) group, and HG + I/R (hyperglycemic + 1 h MCAO and 24 h reperfusion) group. Hyperglycemia was induced in male mice by streptozotocin (STZ) . The blood glucose level was measured with a blood glucose meter 72 h after STZ injection. According to our previous experience, hyperglycemia was designated as a fasting blood glucose level of more than 16.8 mmol/L [In the present study, a total of 54 male WT mice and 58 UCP28 mmol/L .Mice were anesthetized with 2–3% isoflurane during induction, and anesthesia was maintained with 1.5–1.8% isoflurane during the operation. The right middle cerebral artery occlusion (MCAO) model was established using the filament method. Briefly, the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were first separated. Second, a small incision was made in the CCA, and a filament  was inserted into the ICA to block the MCA. Finally, after 1 h of occlusion, animals were anesthetized again, the filament was removed, and reperfusion was performed for 24 h. The mice in the sham group were subjected to the same surgical procedures without blocking the MCA. During the surgery period, the body temperature of the animal was monitored and maintained at 37  ±  0.5 °C with an animal body temperature maintenance instrument . The average body temperature of animal during the operation was quantified. After operation, the animal was put into animal intensive care units  for recovery. The neurological deficit was scored by Zea-Longa’s scale : (0) no 2O for 5 min). Then, the sections were stained with hematoxylin for 4 min. Next, the sections were stained with eosin for 1 min. Finally, the sections were dehydrated and gently covered with coverslip slides. Images were acquired under a high-power field (400 X) with a microscope, and the percentage of karyopyknotic cells was calculated. The cortical area in five brain slices per mouse was quantified.First, the mouse brains were made into paraffin blocks of brain tissue. Then, the brain tissue block was sliced into 4 μm sections using a microtome. The sections were deparaffinized and rehydrated in a gradient of xylene and ethanol  according to the manufacturer’s protocols. Briefly, the sections were deparaffinized and rehydrated in a gradient of ethanol. Then, proteinase K working solution was used to digest the tissues for 30 min at 37 °C. After rinsing, the TUNEL reaction mixture was added to the sections for 45 min at 37 °C. Finally, DAPI was applied to the sections, and the sections were covered with a coverslip. TUNEL-positive cells were observed with a fluorescence microscope, and the percentage of TUNEL-positive cells was calculated.2. The cell medium was replaced per 24 h.The mouse hippocampal neuronal cell line HT22 was originally obtained from ATCC . HT22 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 1% penicillin/streptomycin (HyClone) and 10% fetal bovine serum . The cells were cultured in an incubator at 37 °C with 90% relative humidity and 5% COThe high glucose, genipin and OGD/R groups were as follows: NG group (control treatment of HT22 cells); HG group (high glucose treatment of HT22 cells); NG+OGD/R group (OGD/R treatment of HT22 cells); HG + OGD/R group (OGD/R and high glucose treatment of HT22 cells); NG + OGD/R + GE group (OGD/R and genipin treatment of HT22 cells); and HG + OGD/R + GE group . According to previous studies, the selected high glucose concentration was 50 mM. Glucose was dissolved in PBS to prepare a stock solution and added to the medium at a final concentration of 50 mM. The concentration of glucose in the DMEM that was used was 25 mM. Cells cultured in culture medium containing 25 mM glucose were used as normoglycemic controls.2 and 5% CO2 at 37 °C for 1 h. Finally, the glucose-free medium was replaced with normal medium and reoxygenated for 24 h.First, the cell culture medium was changed to glucose-free medium, and then the cells were placed in a hypoxic incubator  containing 95% NFor genipin treatment, HT22 cells were plated on 96-well plates (5000 cells/well) 24 h before the treatments. After adherence, the cells were treated for 24 h with different concentrations of genipin . At the end of the incubation, cell viability was determined via cell counting kit-8 (CCK-8) .In brief, HT22 cells were plated at 5000 cells/well in 96-well plates. The cells were exposed to different treatments after 24 h of adhesion. After an additional 24 h of incubation, the absorbance was recorded by a SpectraMax reader. The relative intensity of the control group was designated as 100% cell viability, and the corresponding percentages of the experimental groups were calculated relative to the control group.HT22 cells were incubated in culture medium containing 20 μmol/L dihydroethidine (DHE) for 30 min in the different groups, washed twice with PBS, and fixed in 4% paraformaldehyde in PBS for 15 min. Intracellular ROS production was assessed with a fluorescence microscope. Images were captured at 400 X magnification, and the intensity was evaluated by IPP6.0 image analysis software.Mouse brain tissues or HT22 cells were lysed in RIPA lysis buffer containing protease inhibitor cocktails. The protein concentration was measured using a BCA assay kit. Equal amounts of protein (50 µg/well) were loaded into wells for 10% SDS-PAGE, and the resultant gel was transferred to polyvinylidene fluoride membranes. The membrane was incubated with primary antibodies in blocking buffer at 4 °C overnight. The following primary antibodies were used: NLRP3 (1:300), ASC (1:1000), caspase 1 (1:1000), cleavedcaspase 1 (1:1000), UCP2 (1:500), superoxide dismutase (SOD2) (1:1000), TXNIP (1:1500), IL-1β (1:2000), and pro-IL-1β (1:1000). GAPDH (1:1000) and β-actin (1:5000) were used as internal controls for protein loading. Then, the membrane was incubated with secondary antibodies at room temperature for 1 h. Imaging was performed using a BIO-RAD imaging system, and the ratio of the target protein level to GAPDH or β-actin was calculated.The paraffin-embedded brain sections were boiled for antigen retrieval in citrate buffer. Then, the sections were blocked for 20 min with 10% goat serum at room temperature. Next, the sections were incubated with primary antibodies at 4 °C overnight. After being washed three times with PBS, the sections were incubated with secondary antibodies at 37 °C for 1 h, and DAPI was added for 5 min in the dark.HT22 cells from the different groups were seeded on glass slides. The slides of cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The following steps were the same as those described above.The levels of IL-1β and IL-18 in the cell culture supernatant and serum were measured by mouse IL-1β and IL-18 ELISA kits  according to the manufacturer's instructions.p < 0.05.Each experiment was independently repeated a minimum of three times. Statistical analysis was performed with IBM SPSS Statistics version 20.0. All values are expressed as the mean ± standard deviation (SD) or as a percentage of the control. The datasets were analyzed by ANOVA followed by the LSD post-hoc test. Statistical significance was set at −/− animals  in UCP2p > 0.05). Then, the expression of UCP2 was measured by western blotting after genipin treatment. Figure Because the NLRP3 inflammasome is expressed in neurons, we used hippocampal HT22 neurons to explore whether UCP2 deficiency affects the NLRP3 inflammasome activation pathway. Genipin is a specific UCP2 inhibitor that was used to explore the role of UCP2 in HT22 cells. To determine the effect of genipin on the expression of UCP2 and cell viability, we treated HT22 neuronal cells with genipin. First, a dose–response experiment was performed on HT22 cells treated with genipin for 24 and 48 h. Figure To further assess the influence of UCP2 deficiency on HT22 cells exposed to OGD/R and high glucose conditions, we assessed cell viability with a CCK-8 kit. Figure To investigate the role of UCP2 deficiency on NLRP3 inflammasome activation in HT22 cells under OGD/R and high glucose conditions, we measured the expression of NLRP3 inflammasome proteins, including NLRP3, ASC, cleaved caspase 1, caspase 1, TXNIP, and IL-1β, using western blotting and immunofluorescence. Western blotting showed that OGD/R increased the protein levels of NLRP3, ASC, cleaved caspase 1, caspase 1, TXNIP, and IL-1β. Concurrent treatment with genipin further increased the expression of NLRP3, ASC, cleaved caspase 1, caspase 1, TXNIP, and IL-1β in HT22 cells under normal and high glucose conditions. These results suggest that OGD/R activates the NLRP3 inflammasome in HT22 cells. After genipin was added to inhibit UCP2 expression, the NLRP3 inflammasome was further activated. Immunofluorescence analysis confirmed the changes in NLRP3, ASC, cleaved caspase 1, caspase 1 and IL-1β Fig. a. The rep < 0.01). Simultaneous treatment with genipin resulted in obvious increases in OGD/R-induced ROS and MDA production under normal or high glucose conditions (p < 0.01). Manganese SOD (SOD2/MnSOD) is a mitochondria-resident enzyme that functions as the first line in mitochondrial antioxidant defense. We measured the protein levels of SOD2 in the different groups by immunofluorescence and western blotting. The immunofluorescence results showed abundant staining of SOD2-positive neurons among normal HT22 cells. OGD/R obviously reduced the number of SOD2-positive cells. Concurrent treatment with genipin further decreased the immunoreactivity of SOD2 in ODG/R-exposed neurons. Western blot analysis demonstrated that the level of SOD2 protein was lower in the OGD/R group than in the control group, and genipin further decreased the expression of SOD2 compared with that in the OGD/R group under normal or high glucose conditions. These results suggested that UCP2 deficiency enhanced ROS production in HT22 cells under OGD/R and high glucose conditions.Excess ROS production causes different forms of DNA damage, resulting in cell death. We measured ROS production using the DHE probe. As shown in Fig. Our study showed that UCP2 deficiency significantly increased histopathological changes and apoptosis after cerebral I/R in normoglycemic and hyperglycemic mice. Furthermore, UCP2 deficiency enhanced NLRP3 inflammasome activation in neurons in the context of hyperglycemia-exacerbated cerebral I/R damage. Moreover, UCP2 deficiency enhanced NLRP3 inflammasome activation and ROS production in HT22 cells under OGD/R and high glucose conditions. These findings suggest that UCP2 deficiency enhances NLRP3 inflammasome activation and ROS production in neurons in the context of hyperglycemia-exacerbated cerebral I/R damage in vivo and in vitro.UCP2 plays important roles in the process of cerebral I/R damage. The main function of UCP2 may be to stabilize the inner mitochondrial membrane potential and reduce ROS generation . PreviouRecently, the NLRP3 inflammasome has been shown to serve as a critical contributor to cerebral I/R damage . PreviouSome studies suggest that ROS are critical stimuli that activate the NLRP3 inflammasome , 24. ExpIn summary, our data suggest that UCP2 deficiency enhances NLRP3 inflammasome activation and ROS production after neurons in hyperglycemia-exacerbated cerebral I/R damage in vivo and in vitro. UCP2 is likely an upstream mediator of ROS generation and NLRP3 inflammasome activation in hyperglycemia-exacerbated I/R damage. Although the underlying pathological mechanisms behind hyperglycemia-exacerbated I/R damage have not been fully investigated, our study provides a possible novel strategy for the treatment of stroke.


The electrical conductivities of the Bi2Te2.85Se0.15Ix were significantly improved by the increase of carrier concentration. The dominant electronic scattering mechanism was changed from the mixed (T ≤ 400 K) and ionization scattering (T ≥ 420 K) for pristine compound (x = 0.0) to the acoustic phonon scattering by the iodine doping. The Hall mobility was also enhanced with the increasing carrier concentration. The enhancement of Hall mobility was caused by the increase of the mean free path of the carrier from 10.8 to 17.7 nm by iodine doping, which was attributed to the reduction of point defects without the meaningful change of bandgap energy. From the electron diffraction patterns, a lattice distortion was observed in the iodine doped compounds. The modulation vector due to lattice distortion increased with increasing iodine concentration, indicating the shorter range lattice distortion in real space for the higher iodine concentration. The bipolar thermal conductivity was suppressed, and the effective masses were increased by iodine doping. It suggests that the iodine doping minimizes the ionization scattering giving rise to the suppression of the bipolar diffusion effect, due to the prohibition of the BiTe1 antisite defect, and induces the lattice distortion which decreases lattice thermal conductivity, resulting in the enhancement of thermoelectric performance.We investigated the anisotropic thermoelectric properties of the Bi Recentlyectively ,2.23 are well-known high performance thermoelectric materials near room temperature. [zT values in the hot-deformed Bi0.3Sb1.7Te3 (1.3 at 380 K) [0.4Sb1.6Te3 (1.41 at 147 K) [zT values such as hot-deformed Bi2Te2.3Se0.7 (1.2 at 445 K) [2Te2.7Se0.3 (1.10 at 373 K) [2Te2.7Se0.3 (1.13 at 423 K) [2Te2.7Se0.3 with hot-deformation (1.07 at 423 K) [2Te2.7Se0.3 nano crystal (1.31 at 438 K) [2Te2.3Se0.69 (1.2 at 450 K) [1.95Sb0.05Te2.3Se0.7 (zT = 1.3 at 450 K) [P-type and n-type t 380 K) , Te-excet 147 K) , melt-spt 423 K) , CuI-dop7Se0.3 1. at 373 Kt 438 K) , Se-defit 450 K)  and hot-7Se0.3 1. at 423 KzT values of the n-type materials are not compatible with those of the p-type properties because the thermoelectric device’s performance is mainly determined by the average zT values of p-and n-type materials. [2Te3−xSex [Even though the thermoelectric performance of n-type bismuth tellurides has been progressing significantly, the terials.  To reachTe3−xSex ,11,15,16Te3−xSex ,17, I-doTe3−xSex ,17 Ga-doTe3−xSex , CuI-dopTe3−xSex ,19,20, hTe3−xSex ,22,23,242Te2.85Se0.15 compound, synthesized by the ball milling and hot press sintering. The Bi2Te2.85Se0.15 parent compound by zone melting method shows a high zT value (1.1 at 340 K) and an extremely high power factor (~5.5 mW·m−1·K−2 at 300 K) [2Te2.85Se0.15 enhances lattice distortion by the formation of the charge density wave. We propose that the lattice distortion by charge density wave (CDW) suppresses the bipolar diffusion effect and results in the enhancement of thermoelectric performance at high temperature regions. Here, we investigate the iodine doping effects on the polycrystalline Bit 300 K) . Iodine t 300 K) ,17,25,26t 300 K) ,27. From2Te2.85Se0.15Ix, x = 0.0, 0.1, 0.3 and 0.5 mol.%) samples were prepared by melting, ball-milling, and hot-press method. The Bi , Te , Se  elements and BiI3  powder were sealed in evacuated quartz tubes and heated at 1073 K for 24 h with slow cooling to room temperature. A planetary ball-milling pulverized the melted ingot samples for 1 h. The powders were loaded into a graphite die with an inner diameter of 12 mm and sintered by hot-press sintering at 773 K for 5 min under uniaxial pressure of 30 MPa. The relative densities of the hot-pressed samples were above 95% (7.70~7.75 g/cm3) comparing with the theoretical densities (8.02~8.04 g/cm3).The I-doped bismuth telluride (Biσ) and Seebeck coefficient (S) were simultaneously measured under the helium atmosphere by a four-point probe method using a thermoelectric properties measurement system . Thermoelectric properties and X-ray diffraction (XRD) patterns of the hot-pressed samples were performed with the parallel (Pa) and perpendicular (Pe) directions to the press direction. The XRD patterns were obtained using the Cu-κα radiation . The temperature-dependent electrical conductivity (Hn) and the Hall coefficient (HR) were obtained by the relations of H = −1/(RHe)n, where H = ρxy/HR is the Hall coefficient, e is the elementary charge, xyρ is the Hall resistivity, H is the applied magnetic field. The Hall resistivity (xyρ) was measured by the five-probe contact method under sweeping magnetic fields from −5 to 5 T using the physical property measurement system . The Hall mobility was obtained from the relation H = 1/nH eρμ.The Hall carrier concentrations (κ) was obtained by the relation of sλCpκ = ρ, where s, λρ, and pC are sample density, thermal diffusivity, and specific heat, respectively. The thermal diffusivity (λ) was measured by a laser flash method . The sample density was obtained using the sample mass and dimensions. The specific heat (pC) was estimated from high temperature fitting from the measurement data by the physical property measurement system .The total thermal conductivity  compounds for two different planes of parallel  and perpendicular  directions to the press direction, as indicated in the right inset. All the peaks were normalized with the highest (015) peaks. The XRD peaks were indexed with the Bi2Te3 structure  with a small change of lattice parameter.l) peaks, indicating that the bismuth telluride’s quintuple layers were stacked along the parallel direction to the press direction, which was similar to those of the hot-deformed Bi2−xSbxTe2.3Se0.7 [2Te3−xSex nanocrystals [As presented in the left-upper inset, the XRD patterns along the parallel direction showed the enhanced  compounds can be understood by the iodine substitution at the Te sites.The ompounds . The forompounds . Therefoσ(T) of the Bi2Te2.85Se0.15Ix  compounds. The anisotropic measurement of σ(T) for x = 0.0 had two distinctive regions, as shown in σ(T) can be understood by the distinctively different scattering mechanism. The electrical conductivity is written as σ = neµ, where mobility is implicitly represented by the effective mass m* and average relaxation time <τ> by µ = e<τ>/m*. The relaxation time is a function of electronic energy E and temperature T by the relation; r is the scattering factor such that r = −1/2 for acoustic phonon scattering, r = 0 for a neutral impurity scattering, r = 1/2 for an optical phonon scattering, r = 1 for a mixed scattering, and r = 3/2 for an ionized impurity scattering [σ(T) of the pristine Bi2Te2.85Se0.15 compound is attributed to the mixed scattering at a lower temperature than 400 K and the ionized impurity scattering (σ ∝ T1.5) at high temperatures (T ≥ 420 K). The temperature-exponent behavior of Hall mobility may also follow the one of electrical conductivity because the carrier concentration is not sensitive to temperature in a degenerated semiconductor. So, it is reasonable to interpret the scattering exponent behavior from the temperature-dependent electrical conductivity.attering ,31,32,33σ(T) of Bi2Te2.85Se0.15Ix (0.1 ≤ x ≤ 0.5 mol.%) exhibited a metallic or highly degenerated semiconducting behavior, as shown in 2Te2.85Se0.15I0.005 compound (about 1600 S/cm at 323 K) [2Te2.85Se0.15Ix compounds could be regarded as the acoustic phonon scattering [σ ∝ T−1.5, where r = −1.25 (x = 0.1 mol.%), −1.31 (x = 0.3 mol.%), and −1.36 (x = 0.5 mol.%).The t 323 K) . The eleattering ,34 becauH = 1/(neρ)µ were obtained. The Hall carrier concentration (H = n/rHn) and Hall mobility (H = μ/rHμ) were calculated from the following equationsm* is the effective mass of the carrier, Hr is the Hall factor, η = F/kBTE is the reduced Fermi energy, and n(η)F is the n-th order Fermi integral given by [The room-temperature electrical conductivities (left axis) were increased with increasing the iodine doping concentration for parallel (open black square) and perpendicular (closed black square) direction for the press direction, as shown in given by ,35(4)FnThe effective masses of the carrier can be obtained from the Hall carrier concentration, while the reduced Fermi energy is obtained from the Seebeck coefficient as the following equation ,35.(5)Sr = −1/2), the Hall mobilities decreased with increasing Hall carrier concentration, as presented in r = 1), the Hall mobility significantly increased with increasing Hall carrier concentration for perpendicular (solid black line) and parallel (black dotted line) directions, while the Hall mobilities in acoustic phonon scattering decreased with increasing Hall concentration, as shown in In the case of the acoustic phonon dominant scattering (S(T) for the pristine (a) and I-doped Bi2Te2.85Se0.15Ix  compounds (b). The negative Seebeck coefficients indicated the electronic transport of the carrier. The Seebeck coefficients of the pristine sample (x = 0.0) for the perpendicular (−216 µV/K) and parallel (−215 µV/K) directions were similar to the hot-pressed Bi2Te2.85Se0.15 compounds (about −220 µV/K) at room temperature [S(T) of the parent compound was well described by the mixed scattering exponent  below 425 K, while the ionization scattering (r = 3/2) was dominant at a high-temperature range (T ≥ 425 K). This scattering mechanism was also consistent with the S(T) along the Pa-direction, as presented in the inset of perature . The S(TS(T) data for Pe- (closed symbols) and Pa- (open symbols) directions. The solid and dashed lines were a theoretical fitting with the experiment in terms of acoustic phonon scattering (r = −1/2). Therefore, the dominant scattering mechanism was changed from mixed (T ≤ 425 K) and ionization scattering (T ≥ 425 K) in the pristine compound to the acoustic phonon scattering over a wide temperature range in the iodine doped compounds.The temperature-dependent behavior of the Seebeck coefficient on the I-doped compounds (0.1 ≤ x ≤ 0.5 mol.%) showed a different behavior with those of the pristine compound, as shown in −S as a function of Hall carrier concentration Hn at room temperature. Because the mixed (r = 1) and acoustic scatterings (r = −1/2) are dominant in parent and iodine-doped compounds, respectively, at room temperature, we used the corresponding scattering exponent within a single parabolic band assumption. The effective masses of the carriers in the I-doped samples were significantly increased  as compared with the pristine sample . The carrier effective mass could be enhanced by increasing the carrier scattering with the nanoparticles [articles , resonanarticles , Rashba articles ,39, nonparticles , the incarticles ,42 and tarticles , etc. μ and the carrier effective mass m*, the mean free paths λ of the carrier can be calculated by the following equation, as shown in FE is the Fermi energy. The carrier mean free paths λ of the pristine sample Bi2Te2.85Se0.15 are about 10.8 nm (Pe-direction) and 8.0 nm Pa-direction, which are comparable with the zone melted n-type Bi2Te2.79Se0.21 (~7 nm) [2Te2.7Se0.3 (10.82 nm) [λ in perpendicular direction than those in the parallel direction was due to the anisotropic electronic transport along with the in-plane electronic conductivity (perpendicular to the press direction).From the carrier mobility  (~7 nm)  and the 2Te2.85Se0.15Ix were affected by the increase of carrier mean free path rather than the Fermi energy FE , as shown in FE subsequently decreased with the substitution of iodine concentration for x = 0.1 mol.% with the small increase of FE for high doping concentration. The carrier mean free path (λ) can be increased by the preferred crystallographic orientation (texturing effect) in bismuth tellurides [23 compounds included the intrinsic point defects such as the Se vacancy. The Se-deficiency generated the BiTe antisite defect at the Te(Se) sites [2Te2.85Se0.15Ix compounds was attributed to the reduction of point defects.The enhanced Hall mobilities of Billurides  and by tllurides . Since te) sites . The iode) sites . Therefototal (T)κ of the Bi2Te2.85Se0.15Ix  compounds are shown in total(T)κ of the pristine sample (x = 0.0) was increased with increasing temperature (black squares), which is typical behavior of bipolar diffusion effect [total(T)κ of the I-doped samples  decreased with increasing temperature up to the 420 K and then increased again at high temperatures, implying the suppression of the bipolar diffusion effect by iodine doping. The total thermal conductivities of the Pe-direction were higher than those of the Pa-direction by the layer structure of the bismuth telluride [The temperature-dependent total thermal conductivities n effect . The κtoelluride .2Te2.85Se0.15I0.005 compound [2Te2.85Se0.15 are more reliable.However, in contrast with that, the Pe-direction electrical conductivities and Seebeck coefficients of the x = 0.5 mol.% sample were comparable with the data of the Bicompound , the thecompound  were clocompound . Therefoel = L0σTκ), where 0L, σ, and T are the Lorenz number, electrical conductivity, and absolute temperature, respectively. In the case of simple metals, the Lorenz number is 0L = 2/3)·(kB/e)2L= compounds. When we calculated the Lorenz number for the parent compound (x = 0.0) by mixed scattering  and ionization scattering , the Lorenz numbers were about 2.4~2.5 × 10−8 W·Ω·K−2. On the other hand, for acoustic phonon scattering (r = − 1/2), the Lorenz numbers were in the range of (1.6 ~ 2.1) × 10−8 W·Ω·K−2. When we subtracted the electronic thermal conductivity elκ from total thermal conductivity totalκ, the combined contribution of lattice Lκ and bipolar thermal conductivities bκ were obtained, as shown in 2Te2.85Se0.15Ix compounds  even a small amount of the iodine doping. It is generally known that bismuth tellurides have a bipolar effect due to the small band gap. [2Te3−xSex [and gap. ,49 The cand gap. ,49, Cu-dand gap. , CuI-dopand gap. , Bi2Te3−Te3−xSex  as well Te3−xSex . 0σ is a preexponential factor, aE is the activation energy, and Bk is Boltzmann constant [aE and band gap energy gE are obtained by aE ~ 0.076 eV and gE ~ 0.152 eV (not shown), which is estimated by the relation of the band gap energy aE ~ gE/2. In addition, the thermal band gap energy of the Bi2Te2.85Se0.15Ix  compounds is obtained by about 0.14 eV from the gE = maxTmax2eS relation [maxS is the maximum Seebeck coefficient at the temperature (maxT). The estimated band gap energies of the Bi2Te2.85Se0.15Ix  compounds are comparable with the optical band gap (gE = 0.14~0.18 eV) and thermal band gap (gE ~ 0.154 eV) of the I-doped Bi2(Te1−xSex) [2Te3−xIx. [The pristine sample (x = 0.0) showed a semiconducting behavior in electrical resistivity above 400 K. From the Arrhenius equation constant , the actrelation , where Se1−xSex)  and the Te3−xIx.  The obtaTe1 anti-site defects were the most energetically favorable defect in Bi-rich composition. In this case, the minority hole carriers could be generated by the BiTe1 antisite defects in the n-type bismuth telluride [2Te2.3Se0.7−x clearly showed that the bipolar effects became significant with increasing Se deficiencies [2Te2.85Se0.15Ix is supported by the enhanced mean free path of the carriers. The suppression of the bipolar diffusion effect by iodine doping can be described by the inhibition of the vacancy and antisite defects due to stable substitution at the Te-site. The formation energy calculation results showed that the Bielluride . The prielluride ,36. For ciencies . The iodciencies . Therefo2Te2.85Se0.15Ix compounds  clearly showed that the iodine doping decreased the lattice and bipolar thermal conductivities significantly, as shown in It is noteworthy that the lattice and bipolar thermal conductivities 2Te2.7Se0.3 [2Te2.1Se0.9 [The decreased lattice and bipolar thermal conductivity and the unusual anisotropic lattice thermal conductivity can be explained by the lattice distortion (or CDW). Previously, we found the lattice distortion in the hot-deformed CuI-doped Bi2.7Se0.3  and CDW-2.1Se0.9 . The lat2.1Se0.9 ,52,53.2Te2.85Se0.15Ix  compounds were found in the high-resolution transmission electron microscopy (HR-TEM) and electron diffraction (ED) patterns, as shown in 2Te2.7Se0.3 [2Te2.1Se0.9 [The lattice distortion of the Bi2.7Se0.3  and the 2.1Se0.9 . The lat2σ(T)S of the Bi2Te2.85Se0.15Ix  compounds are presented in −1·K−2 for the Pe-direction (1.0 mW·m−1·K−2 for the Pa-direction). The power factors of the I-doped Bi2Te2.85Se0.15Ix  compounds were increased owing to the enhancement of electrical conductivity as compared with the pristine sample. Because of the anisotropic electrical conductivity, the power factor of the Pe-direction was higher than those of the Pa-direction. −1·K−2 at 300 K for x = 0.1 mol.% for the Pe-direction. The power factor of the pristine sample  was much lower than the one of Bi2Te2.85Se0.15 (~2.10 mW·m−1·K−2 at 323 K) [Temperature-dependent power factors t 323 K) .zT values of the Bi2Te2.85Se0.15Ix compounds. The room-temperature zT values with iodine doping concentrations showed that the zT values of the I-doped samples  were higher than those of the pristine sample (x = 0.0) by the enhanced power factor and the decreased lattice and bipolar thermal conductivity. The zT values of the Pe-direction were higher than the Pa-direction from the anisotropic electrical conductivity. The zT value of the x = 0.1 mol.% was reached 0.72 at 423 K for the Pe-direction. Therefore, the Iodine doping suppresses the bipolar diffusion effect and increases the electrical conductivity, resulting in the enhancement of the power factor.rS is the scaled Seebeck coefficient defined by │S│Be/k. 2σS with absolute Seebeck coefficient of the Bi2Te2.85Se0.15Ix  compounds. From the theoretical fitting with experiment, we can estimate the electronic quality factor EB. The electronic quality factor EB values of the Iodine-doped Bi2Te2.85Se0.15Ix  compounds were 5.9 ~ 6.3 mW·m−1·K−2, which was significantly improved from the pristine x = 0 compound (1.3 mW·m−1·K−2) along the perpendicular direction. Because of the anisotropic thermoelectric properties, the EB values of the Pe-direction were much higher than the Pa-direction values. The higher EB values of the I-doped samples as compared to the pristine sample (x = 0.0 mol.%) clearly showed that the I-doping was effective to enhance the thermoelectric performance of the bismuth telluride.A recent study suggested how to find the maximum power factor without experimental optimization by defining the electronic quality factor. The electronic quality factor is defined by (9)BE=S22Te2.85Se0.15Ix  compounds, synthesized by ball-milling and hot press sintering. The anisotropic thermoelectric properties were attributed to the structurally preferred orientation of the bismuth telluride along the (00l) direction with the press direction. The electrical conductivities of the I-doped samples were mainly enhanced by the increased carrier concentration. The Hall mobility was enhanced with increasing carrier concentration due to the increase of the mean free path from 10.8 to 17.7 nm by iodine doping. The enhanced mean free path was caused by the reduction of point defects. We found that the carrier scattering mechanism was changed from the mixed (T ≤ 400 K) and ionization scattering (T ≥ 420 K) for pristine compound (x = 0.0) to the acoustic phonon scattering for iodine doped compounds (x = 0.1~0.5 mol.%). The electron diffraction results clearly showed that the I-doping generated the periodic lattice distortion in the bismuth telluride. For increasing I-doping concentration, the modulation vector in momentum space was increased, in other words, the shorter range of lattice distortion for the higher iodine doping concentration. The periodic lattice distortion could increase the carrier effective mass as the CDW effect. The ionization scattering of the carrier should be minimized to suppress the bipolar diffusion effect in thermal conductivity due to the prohibition of vacancy and antisite defects due to stable substitution at the Te-site, while the lattice distortion decreases the lattice thermal conductivity by iodine doping. Because of the enhanced power factor and the decreased lattice and bipolar thermal conductivity by the iodine doping, we observed the enhancement of zT values from 0.31 (x = 0.0) to 0.56 (x = 0.1 mol.%) at 300 K (0.72 at 423 K for Pe-direction). This research strongly suggests that the minimization of ionization scattering suppresses the bipolar diffusion effect by hindering vacancy and defect states, and lattice distortion decreases lattice thermal conductivity, resulting in the enhancement of thermoelectric performance.In summary, we investigated the anisotropic thermoelectric properties of the Bi


Naturally occurring substances are valuable resources for drug development. In this respect, chalcones are known to be antiproliferative agents against prostate cancer cell lines through various mechanisms or targets. Based on the literature and preliminary results, we aimed to study and optimise the efficiency of a series of chalcones to inhibit androgen-converting AKR1C3, known to promote prostate cancer. A total of 12 chalcones with different substitution patterns were synthesised. Structure–activity relationships associated with these modifications on AKR1C3 inhibition were analysed by performing enzymatic assays and docking simulations. In addition, the selectivity and cytotoxicity of the compounds were assessed. In enzymatic assays, C-6′ hydroxylated derivatives were more active than C-6′ methoxylated derivatives. In contrast, C-4 methylation increased activity over C-4 hydroxylation. Docking results supported these findings with the most active compounds fitting nicely in the binding site and exhibiting strong interactions with key amino acid residues. The most effective inhibitors were not cytotoxic for HEK293T cells and selective for 17β-hydroxysteroid dehydrogenases not primarily involved in steroid hormone metabolism. Nevertheless, they inhibited several enzymes of the steroid metabolism pathways. Favourable substitutions that enhanced AKR1C3 inhibition of chalcones were identified. This study paves the way to further develop compounds from this series or related flavonoids with improved inhibitory activity against AKR1C3. AKR1C3 is a monomeric enzyme of 37 kDa, displaying a TIM-barrel structure with a characteristic (αβ)8-barrel motif [Aldo–keto reductase 1C3 (AKR1C3), also called 17β-hydroxysteroid dehydrogenase (17β-HSD) type 5, belongs to the large superfamily of aldo–keto reductases which is divided into 15 subfamilies. In humans, AKR1C3, together with AKR1C1, AKR1C2, and AKR1C4, constitutes the AKR1C family of hydroxysteroid dehydrogenases (el motif .AKR1C3 is a NAD(P)H-dependent enzyme catalysing the reduction of endogenous substrates bearing aldehyde and ketone functions into their hydroxyl counterparts that can then undergo further conjugation reactions such as sulphation and glucuronidation [AKR1C3 is a key enzyme in steroid hormone metabolism . In its The enzyme also has 20α-hydroxysteroid dehydrogenase activity , catalys2 synthase [2 to PGF2α and PGD2 to 9α-11β-PGF2α [2α metabolites are able to bind to the prostaglandin receptor, inducing proliferative and inflammatory processes and thereby preventing differentiation and apoptosis [Moreover, AKR1C3 also plays a role in prostaglandin (PG) metabolism and is known as PGFsynthase . The enz1β-PGF2α ,15. Bothpoptosis ,17.2α levels in diseased tissues, thereby promoting proliferation processes. On the other hand, with its ability to reduce and deactivate drugs that carry carbonyl groups, AKR1C3 contributes to the exacerbation of disease progression. The process of drug inactivation by carbonyl reductases is part of the mechanisms of drug resistance and is a major threat to cancer therapy efficacy [As regulators of hormone signalling, steroid-metabolising enzymes are important in several biological processes, e.g., sexual development, reproduction, and maintenance of energy level or response to stress, and changes in their enzymatic activities can lead to serious diseases. Indeed, AKR1C3 is associated with several diseases such as prostate cancer , breast efficacy . For exaefficacy ,27. Sincefficacy .Many steroidal or non-steroidal AKR1C3 inhibitors have already been developed and characterised ,25,29. MAmong the various strategies used to identify promising active scaffolds or derivatives, exploring the therapeutic potential of natural products has led to major contributions and breakthroughs, especially in the field of cancer therapies. Since 2012, Newman and Cragg have deeply highlighted the importance of natural products and all the structurally related analogues in the drug discovery processes .50 of 11 µM and 13 µM, respectively [50 generally ranging from 2 to 50 µM, except baccharin which showed a higher potency, with an IC50 of 100 nM [50 between 5 µM and 20 µM, and the compounds with the highest selectivity over other AKR1C enzymes had an IC50 of around 13.5 µM [50 values from 300 nM to 50 µM were observed in studies analysing the effect of dietary flavonoids on AKR1C3 [50 values of 7.7 µM and 29 µM [50 below 50 µM. In terms of structural considerations, in this former study, five chalcones were either mono- or di-hydroxylated, and three showed a backbone bearing a phloroglucinol moiety.Various natural polyphenols, such as flavonoids or alkaloids, have shown significant inhibitory activities against AKR1C enzymes ,34,35. Cectively . Moreoveectively ,40. Cinnf 100 nM ,42. More 13.5 µM . IC50 van AKR1C3 ,45. In and 29 µM . In anotnd 29 µM . Only th18–29), along with isoliquiritigenin (16) and butein (17) obtained from commercial sources. Our goal was to better understand the AKR1C3–chalcone interaction and to develop an efficient chalcone-based AKR1C3 inhibitor.Our current research aimed to further explore and clarify the importance of the substitution pattern on the inhibitory activity against AKR1C3 for a set of 12 synthetic chalcones  and butein (17); R6′ = OH for 19–23, 26, and 29; R6′ = OCH3 for 18, 24, 25, 27, and 28) in combination with hydrophilic or lipophilic substituents  at C-2, C-3, and/or C-4 positions  was the most active derivative against AKR1C3, followed by analogues 19 and 20, with IC50 values in the range of 2 µM. Chalcone 21 was significantly less active than the previous three derivatives with an IC50 of 5.18 µM. The weaker inhibition potency of chalcone 18 was reflected in the higher IC50 of 11.91 µM. Compound 25 ran into saturation already at low concentrations without reaching the saturation level of 100%, so a determination of the IC50 value was not performed.To further analyse structure-activity relationships, ICMF-11 in ) which w19, 20, 21, and 23, alongside weak inhibitors 18 and 25. The results are shown in The influence of the chalcone derivatives on the activity of other human steroid hormone-metabolising enzymes was investigated in order to determine the selectivity of the compounds. To this end, the three other members of the AKR1C family were examined, along with several members of the 17β-HSD family, i.e., enzymes capable of catalysing the conversion of the same or similar substrates as AKR1C3. Screening at 10 µM compound concentration included the four strong AKR1C3 inhibitors 23, showed no selectivity with respect to the enzymes of the ARK1C family. This derivative also significantly inhibited HSD17B2 (88.8% at 10 µM) and to a lower extent HSD17B1 and HSD17B3, with 53.3% and 68.6% inhibition, respectively. Chalcones 19, 20, 21, 18, and 25 were weak inhibitors of AKR1C2, as they inhibited not more than 42.3% of the enzyme’s activity at 10 µM . Among the 11 enzymes considered in the current screening, AKR1C1 was the only target for 18 and 25, with inhibition values of 96.5% and 87.9%, respectively, at 10 µM concentration  but removed before chalcone docking. The resulting docking poses for chalcones were evaluated by ranking the docking scores  and by iThe binding site of AKR1C3 has been described as consisting of a main binding point at amino acids Tyr55 and His117 which are part of the catalytic tetrad  . MoreoveIn the course of this study, we aimed to find new effective inhibitors for the human enzyme AKR1C3. We focused on chalcones as the central core and tried to identify key substitution patterns that can enhance inhibition of AKR1C3.18, isolated from the plant Melodorum fruticosum, was previously identified as a mild inhibitor of AKR1C3 , two other natural phenolic chalcones, isoliquiritigenin (16) and butein (17), also included in the study, were less active . From a structural perspective, several changes occurred in the substitution pattern of these three derivatives. First, in order to explore the importance of the methoxy substituents at the C-6′ and C-4 positions of ring A and ring B, respectively, chalcones 19  were prepared as phenolic analogues of 18 (R4 = R6′ = OCH3). This led to a loss of the inhibitory potential for 24 (24.6% inhibition at 10 µM) but a significant gain for 19 with 78.1% inhibition at 10 µM. Meanwhile, pentaphenolic chalcone 29 was as active as 18 at the same concentration.Chalcone   and con20–22, 26) or a methoxy group (27 and 28). The latter analogues of 18 bearing electron-rich substituents in C-4 position (R4 = Cl for 27 or R4 = F for 28) were both inactive at 10 µM . Corresponding compounds 22 (R4 = Cl) and 26 (R4 = F), with a phenolic function at the C-6′ position, were threefold more potent, with 70.8% and 68.5% inhibition of AKR1C3 activity at 10 µM. Two other chalcones bearing a methyl group on ring B either at the C-3 or the C-4 position, chalcones 20 and 21, were prepared and evaluated. They both exhibited a higher inhibitory potential at 10 µM than all the chalcones mentioned before, with 89.2% and 87.9% inhibition.A series of derivatives with lipophilic substituents on the C-4 position were prepared and could be divided into subgroups depending on the nature of the C-6′ substituent, which was either a phenol function  exhibited 90.9% inhibition at 10 µM, being threefold more efficient than the corresponding methoxy analogue 25 . All these results showed the importance of the nature of the C-6′ substituent with the phenol function highly favourable for the best inhibitory potential. This parameter has to be combined with the substitution of the B ring by lipophilic atoms or groups. This B ring can be mono- or di-substituted without any impact on the inhibitory potential of the corresponding analogues. A similar trend was reported in a study comparing the inhibitory effect of 2′-hydroxy- and 2′-methoxychalcone on AR-dependent transcription in androgen-dependent LNCaP cells, with the phenolic derivatives being generally more active than or as active as their methoxylated counterparts [As the AKR1C3-binding site can accommodate larger inhibitors , 2,4-dicterparts .19, 20, 21, and 23) along with 18, as the hit compound, and 25, as a weak inhibitor. The 2,4-dichloro-2′,4′,6′-trihydroxychalcone 23 showed the most promising IC50 of 1.08 ± 0.27 µM . Interestingly, shifting the methyl group from the C-4 position (21) to the C-3 position (20) led to an inhibitor almost threefold more potent, although both compounds displayed comparable inhibition in the previous screening at 10 µM  all showed a similar orientation within the binding pocket of the protein  and naringenin chalcone (29), in our study. Using placental microsomes and compound concentration of 10 µM, the authors reported 17β-HSD inhibition of 10% (16) and 2.4% (29), respectively. This is less than what was measured in our study, with 16 and 29 reaching 31.1 ± 3.2% and 40.6 ± 7.7% inhibition of AKR1C3, respectively. However, results cannot be compared directly because the assay setups differed, and unlike in our assay using bacterially overexpressed AKR1C3, an undefined mixture of 17β-HSDs was analysed with the placenta microsomes. In total, 10 17β-HSDs can be found in the human placenta [In the search for AKR1C3 inhibitors, many steroidal and non-steroidal inhibitors have been isolated or generated and analysed ,29. Amonplacenta  would ha50 analyses. AKR1C2 was inhibited as well by all compounds, albeit to a lesser extent. Only chalcone 23, the best inhibitor of our chalcone series, strongly inhibited this enzyme. The low selectivity of the compounds is not very surprising, because it is very difficult to develop specific AKR1C isoform inhibitors due to the high homology (>84% of amino acids) between the human AKR1C enzymes, the very similar structures, and overlapping substrate preferences [In addition to inhibitory potency, selectivity is an important feature in drug development. Selectivity analyses were thus performed by screening the most potent AKR1C3-inhibiting chalcones on putative off-targets, including the other enzymes of the AKR1C family and several 17β-HSDs. The activity of enzymes AKR1C1 and AKR1C4 was inhibited effectively by nearly all tested compounds at 10 µM. The inhibition seemed to be even stronger than that for AKR1C3, but this would have to be verified by ICferences ,34. Indeferences ,45. Chalferences . Bearingferences  can be tIt would be undesirable to inhibit the other isozymes of the AKR1C family when designing AKR1C3 inhibitors. AKR1C2 plays a key role in the inactivation of DHT to the non-potent AR ligand 3α,17β-androstanediol in the prostate . Additio18–21 and 23, which efficiently targeted AKR1C3, also inhibited the steroid-metabolising 17β-HSDs of the short-chain dehydrogenase reductase family, although not as strongly as AKR1C enzymes. HSD17B1 converts estrone to oestradiol [Chalcones stradiol  and trigstradiol . HSD17B2The six chalcones evaluated for selectivity were nearly ineffective on the SDR enzymes HSD17B4, HSD17B7, HSD17B10, and HSD17B14 C. Those 50 of 2.6 µM by naringenin chalcone (29) [In the future, the panel of off-targets should be expanded. For example, Le Bail et al. showed that aromatase in human placental microsomes was inhibited with an ICone (29) . Moreoveone (29) . Thus, bone (29)  should b19, 23, or 25. Whether this was due to the proliferative effects of the compounds on HEK293T cells or experimental variation is not clear. Surprisingly, chalcone 25, a weak AKR1C3 inhibitor that could not be docked into the binding pocket of AKR1C3, had cytotoxic effects in almost the same concentration range as chemotherapeutic agents cisplatin and etoposide, conventionally used in cancer treatment. The underlying mechanism is unclear and would have to be analysed in future studies.In view of cancer treatment, cytotoxicity to target cells in addition to inhibition of AKR1C3 would be a valuable dual activity. However, normal cells should be as little affected as possible. The strong AKR1C3 inhibitors of our study were found to be cytotoxic only at high concentrations, if at all, to HEK293T cells which we took as a representative cell line for non-malignant cells. Interestingly, cell viability appeared to be higher than 100% at some concentrations when cells were treated with compounds https://www.uniprot.org, accessed on 5 January 2022)). Indeed, low endogenous expression was the reason to use this cell line for over-expression of AKR1C enzymes for inhibition screenings. Additionally, the HSD17B enzymes included in our screens were most probably not responsible for the cytotoxic effects. HSD17B2 and HSD17B3 are not present in HEK293 cells, and the weakly expressed HSD17B1 was not inhibited by 25, the chalcone with the highest toxicity. Cytotoxicity of 25 can also not be explained by inhibition of HSD17B7, an enzyme expressed ubiquitously and needed for endogenous cholesterol synthesis, because impaired endogenous cholesterol supply should have been compensated by excess exogenous cholesterol in the culture medium. Thus, most probably, other targets in the cells are affected by the chalcones.Although tested chalcones showed strong inhibition effects on the enzymes of the AKR1C family, the observed cytotoxicity of HEK293 cells cannot be attributed to this inhibition, because AKR1C enzymes are either absent or only negligibly expressed in HEK293 cells  in deuterated solvents and calibrated using the residual undeuterated solvent resonance as an internal reference. Chemical shifts δ are given in ppm and coupling constants J in Hz. Mass spectrometry analyses were performed on a JMS-700  double-focusing mass spectrometer with reversed geometry, equipped with a pneumatically assisted ESI source. The reactions were monitored by analytical thin-layer chromatography using Polygram Sil G plate . Column chromatography was performed by using silica gel 60 Å (particle size 40−63 µm) from Fisher Scientific. Flash chromatography purifications using prepacked columns  were carried out on a CombiFlash Rf-200 apparatus equipped with a gradient pump, a column station with a DASi introduction system, a multiwavelength UV detector, a fraction collector, and appropriate software to control the device . Isoliquiritigenin (16) and butein (17) were purchased from Aldrich .All solvents were dried and distilled before use. Reactions were performed in an inert nitrogen atmosphere. Unless otherwise stated, materials purchased from commercial suppliers were used without further purification. 1 or 2 (1 mmol) in EtOH (3 mL) were added a selected benzaldehyde (1 mmol) and KOH (10 mmol). The reaction mixture was stirred at room temperature for 48 h, and then, ice-cold 10% aqueous HCl solution (20 mL) was added. Unless otherwise stated, the resulting mixture was extracted with EtOAc (3 × 10 mL), and combined organic layers were dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel eluted with the appropriate mixture of solvents to afford the corresponding chalcone.Method A: To a stirred solution of acetophenone 1 or 2 (1 mmol) in dioxane/water  were added a selected benzaldehyde (1 mmol) and NaOH (10 mmol). The reaction mixture was stirred at room temperature for 24 h, and then, ice-cold 10% aqueous HCl solution (20 mL) was added. The resulting mixture was extracted with EtOAc (3 × 10 mL), and combined organic layers were dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel eluted with the appropriate mixture of solvents to afford the corresponding chalcone.Method B: To a stirred solution of acetophenone Method C: To a stirred solution of protected chalcone (1 mmol) in MeOH (30 mL) was added concentrated HCl (1.5 mL). The reaction mixture was stirred at room temperature for 40 h and then diluted with water (30 mL) and extracted with EtOAc (3 × 20 mL), and combined organic layers were dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with the appropriate solvent to afford the desired products.p-toluenesulphonic acid monohydrate (10 mmol). The reaction mixture was stirred at 60 °C for 1 h and concentrated under reduced pressure. The mixture was then diluted with water (10 mL) and a saturated aqueous solution of NaHCO3 (10 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with the appropriate solvent to afford the desired products.Method D: To a stirred solution of protected chalcone (1 mmol) in DCM/EtOH  at 0 °C was added Further information on the synthesis of compounds of this study can be found in E. coli BL21 DE3 or E. coli BL21 DE3 Codon Plus RP  or in HEK293 cells as described in [For enzymatic assays and inhibitor testing, all enzymes were cloned into bacterial or mammalian expression vectors. AKR1C3 and the human hydroxysteroid dehydrogenases HSD17B1, HSD17B2, HSD17B4 (SDR domain), HSD17B7, and HSD17B10 were cloned into a pGEX vector as described in ,72. The ribed in ,72,73. B3H-labelled steroid , and chalcone dissolved in DMSO  in a final concentration of 10 µM for screenings or 0.1 µM to 50 µM (strong inhibitors) and 0.5 µM to 100 µM (weak inhibitors) for IC50 determination . For a detailed assay setup, see v:v) after the time needed to convert approximately 30% of the substrate in a control assay with 1% DMSO, without inhibitor candidate. Substrates and products were extracted from the reaction mixture by solid-phase extraction using C18 cartridges  and separated by RP–HPLC in a Beckman–Coulter HPLC Gold system, using the Luna 5 µm C18(2), 125 × 4.0 mm column (Phenomenex). The mobile phases used were H2O:ACN (57:43 (v:v); for assays of estrone (E1) to oestradiol (E2), E2 to E1, and ∆4-androstene-3,17-dione to testosterone conversion), H2O:ACN (49:51 (v:v); for assays of progesterone to 20α-hydroxyprogesterone conversion), and H2O:MeOH (35:65 (v:v) for assays of dihydrotestosterone (DHT) to 3α-androstanediol conversion) at a flow rate of 1 mL/min. Radioactivity was detected by online scintillation counting by using a radioactivity monitor . Conversion of substrates to products was determined by integration of the peak area of the substrate and product peaks in percent. For calculation of inhibition, the conversion of control assay (assay without inhibitor) was set to 0% inhibition. All assays were run in triplicates, and mean values ± SD are reported.Inhibition assays were carried out as described in ,74. Brie50 values, percent inhibition values were fitted using the Enzyme Kinetics module of SigmaPlot 13.0 and the one-site ligand-binding model according to the formula f = Bmax * abs(x)/(Kd + abs(x)).For the determination of IC2 in constant humidity. Before cell seeding, culture flasks and multiwell plates were coated with collagen G .HEK293T cells were purchased from ATCC (CRL-3216). For the cultivation of HEK293T cells, Dulbecco’s modified Eagle medium  supplemented with 10% FCS (Pan Biotech GmbH), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pan Biotech GmbH) was used. The cells were cultured at 37 °C and 5% CO® Blue (CTB) Cell Viability Assay was used . A total of 10,000 cells in a 200 µL culture medium were seeded into each well of a 96-well plate. Then, 24 h after seeding, the cells were treated with 0, 1, 2.5, 5, 7.5, 10, 15, 25, 50, or 100 µM of the different compounds (3 wells per compound and concentration) and incubated for an additional 72 h. For measurement of cell viability, 20 µL of CTB reagent were added to each well, and after 5 h at 37 °C, the metabolic activity was quantified by recording the fluorescence signal  on a Spark plate reader . Data are shown as mean values ± standard error of the mean (SEM). Statistical significances were calculated by one-way ANOVA (Dunnett’s multiple comparison test), * p < 0.05, ** p < 0.005, *** p < 0.0002, **** p < 0.0001.For studying the cytotoxicity of the different chalcones and the positive control compounds cisplatin and etoposide on HEK293T cells, the Cell Titerwww.inteligand.com, accessed on 10 September 2021). All compounds were prepared for docking by energetically minimising them with omega 2.5.1.4.  [Molecular docking simulations were carried out on the crystal structure of the enzyme AKR1C3 co-crystallised with inhibitor 3-phenoxybenzoic acid  in GOLDNM, USA) ,77. DuriNM, USA) .18 allowed the identification of various substituents that could be linked to a significantly improved inhibition of AKR1C3 activity. Among them, chalcones bearing a phloroglucinol ring showed better potency than their corresponding C-6′ methoxylated analogues. On ring B, lipophilic substituents were well tolerated, while the presence of a phenol function at the C-4 position systematically led to inactive derivatives. Substitution with OCH3, CH3, Cl, or F could be either in position C-4 or positions C-2 and C-4 of ring B. Combination of the optimal substitution parameters led to the identification of 2,4-dichloro-2′,4′,6′-trihydroxychalcone 23, 10-fold more active than the natural chalcone 18, the hit derivative from a previous study.In the current study, structural optimisation of the hit backbone of natural chalcone 23 also showed that interaction with Tyr319 could be strengthened with bulkier substituents on ring B. Therefore, the current study paved the way to a new in silico design of chalcones, which could be led as a fragment growing approach starting from 23, to further explore and develop AKR1C3 selective and efficient inhibitors.Docking poses of the most active chalcones confirm the importance of the C-6′phenolic function. These results bring new insights into the development of AKR1C3 inhibitors. Nevertheless, selectivity among the AKR1C family has not been successfully addressed. This is a major concern, and efforts are still required. Docking poses of


Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia.In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP).Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis.We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia.  Myocardial ischemia is a severe pathological condition that mediates the progression of multiple cardiovascular diseases, including myocardial infarction, stroke, and peripheral vascular disease . An earlAdaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is an important intracellular binding partner for Adiponectin Receptor (AdipoR) contributing to the pathogenesis of acute injury or fibrosis ,13. PrevEight-week-old C57BL/6 mice  were anesthetized by injection of sodium pentobarbital  followed by tracheal intubation aided by a rodent ventilator. The mice were randomly divided into two groups: control (n = 6) and I/R (n = 6). By a standard surgical approach, ischemia-reperfusion injury (IR) was induced by 30 min of ischemia followed by 6 h of reperfusion injury . Sham su2, 5% CO2, and 1% O2) and maintained at 37°C for 4 h [To establish a cellular hypoxia/reperfusion model (H/R), serum/glucose-free DMEM was used. H9C2 cells (rat embryonic-heart derived cell lines) in H/R group, obtained from the American Type Culture Collection, were cultured in anaerobic conditions -APPL1 , the scramble siRNA plasmids overexpressing APPL1  and its control  were obtained from Shanghai GenePharma Co., Ltd. . The si-APPL1 (50 nM) and si-NC (50 nM) were transfected into H9C2 cells using Lipofectamine 2000  according to the manufacturer’s instructions. After 24 h, the cells were subjected to H/R treatment.﹣ΔΔCt method [The total RNA was extracted from heart tissues or H9C2 cells with Trizol reagent . After authentication of RNA quality and quantity, total RNA was reversely transcribed into complementary DNA using a Transcription kit  according to manufacturer’s protocols. A SYBR green qPCR assay kit  was employed to amplify the expression level of APPL1 with GAPDH as the internal references. The PCR conditions consisted of predegeneration for 5 min at 95°C, and cycles of 40 at 95°C for 10s and at 60°C for 30s. The 2t method  was adopThe heart tissues and H9C2 cells of each group were lysed with RIPA lysis buffer  to extract the total protein. Then, 25 µg protein samples were fractionated by 10% SDS-PAGE and transferred onto PVDF membranes. Thereafter, the membranes were blocked, incubated at 4°C overnight with primary antibodies, and corresponding HRP‐labeled secondary antibody at room temperature for 2 h. Primary antibodies, including anti-APPL1 antibody (1:1000), anti-NF-κb p65 antibody (1:1000) and anti-Cox2 antibody (1:1000), anti-Bcl-2 antibody (1:1000), anti-Bax antibody (1:1000), anti-Cleaved PARP antibody (1:500), anti-APAF1 antibody (1:1000), anti- Cleaved caspase9 antibody (1:1000), anti-Caspase9 antibody (1:1000), were obtained from Cell Signaling Technology. Chemiluminescent (Immobilon ECL Ultra Western HRP) was used to develop color (Sigma-Aldrich) models. The protein bands were visualized using an automatic gel imaging analyzer. The intensity of the bands was quantified using Image Lab software and normalized to GAPDH expression.The heart tissues were fixed with 4% neutral paraformaldehyde, washed with running water for 5 min after 48 h, dehydrated with gradient alcohol, and sliced into 3–4 sections (5 μm thick) using the paraffin slicing machine. Subsequently, the tissue sections were baked at 70°C for 60 min, followed by deparaffinization, hydration, HE staining, and sealed with neutral balsam. After HE staining, a light microscope was employed for histological observation.3/well. After 24 h of incubation, 10 μL of cell counting kit-8 (CCK-8) reagent was added to each well and cultured at 37°C for 1 h. The absorbance of each sample was measured at 450 nm using a Microplate Reader . This experiment was repeated at least three independent times.H9C2 cells were plated into 96-well plates at a density of 5.0 × 10Transfected H9C2 cells were collected for detecting activities of lactate dehydrogenase (LDH) following the manufacturer’s instructions of LDH cytotoxicity assay kit . Additionally, the concentrations of inﬂammatory cytokines including TNF-α, IL-1β and IL-6 in supermatants of cells were determined by specific ELISA kits .To assess apoptosis rate of H9C2 cells, 4% paraformaldehyde was used to fix the cells. PBS containing 0.3% Triton X-100 was added to H9C2 cells. Finally, TUNEL solution (50 µl) was added for staining, and nuclear cells labeled positively were considered as apoptotic cells. The positive cells were detected by a fluorescence microscope  at least five random separate fields. The percentage of apoptosis cells was calculated and analyzed.https://www.string-db.org/). Next, IP assay was used to explore their association. Cells were collected and lysed with IP lysis buffer containing protease inhibitor. After centrifugation at 12,000 × g at 4°C, anti-APAF1 or Caspase9 antibody (1 µg) was added into the supernatant on a rotating platform overnight at 4°C. Subsequently, 50 µl of SureBeads™ protein G magnetic beads  were added into the above mixture at 4°C with gentle rotation for 4 h. The pellets were dissolved in 60 µl 1x electrophoresis sample buffer and boiled at 95°C for 5 min. Samples (30 µl) were analyzed by western blotting as aforementioned.In order to further explore the mechanism of accelerating the inflammatory injury and apoptosis of cardiomyocytes after APPL1 silencing, it was predicted that APPL1 could combine with caspase9 /APAF-1 through String database . One-way ANOVA followed by turkey’s post hoc test was performed to determine the differences in the means among multiple groups. To explore the role of APPL1 in myocardial ischemia/hypoxia-reperfusion injury, the APPL1 expression was measured by RT-qPCR and western blotting. As shown in To investigate the role of APPL1 in H9C2 cells under H/R exposure, the APPL1 knockdown was achieved by transfection of siRNA-APPL1. In To further investigate the role of APPL1 in H9C2 cells challenged with H/R,, the effect of APPL1 knockdown on inflammation and cell apoptosis was analyzed. The inflammatory cytokines including TNF-α, IL-1β and IL-6 were measured with ELISA kits. As shown in To investigate the detailed mechanism underlying the role of APPL1 in H9C2 cells subjected to H/R treatment, the interaction in APPL1 and caspase9/APAF-1 were predicted using String database. The western blotting results demonstrated that APPL1 knockdown enhanced the expressions of APAF-1 and Cleaved caspase9 in H9C2 cells with H/R treatment . Next, tAPAF1 inhibitor reversed the effect of APPL1 knockdown on the cell viability, LDH secretion, inflammation and apoptosisTo confirm whether APAF-1/Caspase9 signaling pathway mediated the role of APPL1 in H9C2 cells exposed to H/R,, APAF1 inhibitor (ZYZ-488) was used in further study. As shown in Myocardial ischemia-reperfusion (I/R) injury is a major stimulative factor for the morbidity and mortality related to coronary artery diseases . EmerginIn our study, the APPL1 expression was significantly reduced in cellular H/R model and the myocardial tissue of I/R injury mice model, suggesting APPL1 was closely related to pathogenesis of myocardial ischemia. Lactate dehydrogenase (LDH) level was remarkably increased at the end of ischemia and continued to be at aberrantly high levels throughout reperfusion progression . LDH hasTo investigate the detailed mechanism underlying the role of APPL1 in myocardial ischemia, the interaction between APPL1 and caspase9/APAF-1 was predicted and demonstrated by our findings. Furthermore, APPL1 exerted negatively regulative effect on the expressions of APAF1 and cleaved caspase9, which implied that APAF-1/Caspase9 signaling pathway may mediate the role of APPL1 in myocardial ischemia. It has been reported that APAF-1 was involved in the protective effect of caspase recruitment domain-containing protein 9 on cardiomyocytes from apoptosis following myocardial I/R injury in vivo and in vitro . The APATaken together, APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis, which was, to our interest, abrogated by APAF1 inhibitor. Thus, APPL1 inhibits myocardial I/R injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be presented as a novel and effective target for the treatment of myocardial ischemia.