Giardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated.G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome , the secretion levels of IL-1β, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1β secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice.Recombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1β secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice.Alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1β and pro-caspase-1, significant enhancement of IL-1β secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.Overall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease  Giardia duodenalis, an extracellular protozoan parasite, colonizes the small intestines and leads to 280 million cases of giardiasis annually, with infection accompanied by diarrhea, especially in young children in developing countries [G. duodenalis cysts, after which the cysts enter the stomach and are subjected to excystation by gastric acid. Giardia duodenalis trophozoites attach to the duodenal epithelial cells and trigger nausea, vomiting, diarrhea, abdominal pain and weight loss. Individuals with immunodeficiency and cystic fibrosis can be easily infected. Infection can also occur through oral and anal sex [G. duodenalis infections [G. duodenalis infections.ountries . People fections . Howeverfections . ConsequNeospora caninum [Paracoccidioides brasiliensis [Leishmania spp. [G. duodenalis triggers intracellular NLRP3 inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs) [G. duodenalis infection in vivo remains to be determined.Inflammasomes, a class of cytosolic complexes of proteins, are part of the innate immune response that aid in the defense against invasion by pathogenic microorganisms and mediate inflammatory responses . Among t caninum , Paracociliensis  and Leisnia spp. , but thenia spp. . Based oes (EVs) . HoweverG. duodenalis and play vital roles in trophozoite movement in the small intestine and in the attachment to epithelial cells. To better adapt to its living environment and enhance its pathogenicity, G. duodenalis trophozoites develop unique cytoskeletal structures composed of eight flagella, one median body and one ventral disc [Giardia duodenalis trophozoites use their cytoskeleton to move through the upper small intestine, especially the duodenum, and attach to the intestinal epithelium. They continually migrate and re-attach to epithelial cells as well as using the metabolism of cells. Thus, there is a tight connection between their cytoskeletons and virulence. Giardia duodenalis-specific giardins are components of cytoskeletal structures [G. duodenalis-triggered diarrhea, alpha-giardins are highly expressed and display immunoreactivity during infection [G. duodenalis trophozoite motility and growth rate [G. duodenalis [G. duodenalis itself [G. duodenalis infection, as well as their underlying mechanisms, remains unknown.Giardins were first described as structural cytoskeleton components of ral disc . Giardiaructures  and are ructures . They canfection . Alpha-1nfection . Alpha-8wth rate . Alpha-1odenalis . Alpha-1s itself . HoweverG. duodenalis pathogenicity, explored whether alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vivo and determined the roles of these two giardins in G. duodenalis pathogenicity using mice as a model. Our overall aim was to develop promising targets for the prevention of G. duodenalis infection.In the present study, the recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin and pcDNA3.1(+)-alpha-7.3 giardin were transfected into primary mouse peritoneal macrophages, and the targets that activated the host NLRP3 inflammasome were subsequently screened. We also evaluated the roles of the NLRP3 inflammasome in Female wild-type (WT) C57BL/6 mice, aged 5-8 weeks, were purchased from LiaoNing Changsheng Experimental Animal Center . Mice were given water ad libitum, fed a sterilized diet and housed under a 12/12-h light/dark cycle. Prior to infection, the mice were given antibiotics ad libitum in drinking water supplemented with ampicillin (1 mg/ml), vancomycin (1 mg/ml) and neomycin (1.4 mg/ml)  . Mice thG. duodenalis trophozoites  were cultured in TYI-S-33 medium supplemented with 12.5% fetal bovine serum  and 0.1% bovine bile  under microaerophilic conditions. Confluent trophozoites were harvested on ice and passaged at a ratio of 1:4 for further proliferation.The WB Giardia duodenalis cysts were induced as previously described [6 trophozoites/ml with pH 7.1 induction cyst formation medium (modified TYI-S-33 medium with 0.05% bile concentration). The trophozoites were cultured under anaerobic conditions at 37 °C until the logarithmic stage. The culture medium was replaced with induction cyst formation medium , and G. duodenalis was cultured at 37 °C for 48–96 h, during which cyst formation was observed under the microscope. After most trophozoites had been induced to form cysts, the culturing mixture was collected and re-suspended in sterile deionized water to lyse the remaining trophozoites. The cysts were counted and preserved at 4 °C for subsequent mouse gavage assays.escribed , and theGiardia duodenalis extracellular vesicles (GEVs) were enriched as previously described [6 parasites/ml and cultured for 12 h. GEVs were separated from the culture supernatants by successive centrifugation at 2000 g for 10 min, 10,000 g for 45 min and 100,000 g for 60 min. The pellets were dissolved in phosphate-buffered saline (PBS), quantified using a BCA protein assay kit  and stored at − 80 °C or directly used for further assays.escribed . Trophozg for 10 min. The harvested cells were detected using the CD11b marker by flow cytometry until the cell purity was > 98%, then added to 6-well cell culture plates (4.5 × 106 cells/well) and cultured in RPMI 1640 medium  supplemented with 10% FBS  at 37 °C and 5% CO2.Primary mouse peritoneal macrophages were prepared as previously described . Briefly7 trophozoites in 1 ml of TRIzol reagent , genomic DNA was extracted from G. duodenalis total RNA using MonScript dsDNase  and complementary DNA (cDNA) was synthesized using the MonScript RTIII Super Mix (Monad) according to the manufacturer’s instructions.RNA was extracted from 1 × 10G. duodenalis was obtained from the NCBI GenBank. Specific seamless cloning primers were designed separately for each target gene using Primer 5.0. The forward primers (5ʹ− 3ʹ) were composed of three parts, namely the overlap sequences with EcoRV-linearized pcDNA3.1(+) vector (TGGTGGAATTCTGCAGAT) and the initiation codon ATG and GNN (if the first base of the target gene was not G). This was done to enhance the expression efficiency. Additionally, no fewer than 16-bp combinative bases  were included. The reverse primers (5ʹ− 3ʹ) were composed of two parts, namely the overlap sequences with EcoRV-linearized pcDNA3.1(+) vector (GCCGCCACTGTGCTGGAT) and no fewer than 16-bp combinative bases . The primer sequences are listed in Table The CDS sequence information of the target genes of G. duodenalis cDNA as the template. The eukaryotic expression vector plasmid pcDNA3.1(+) was linearized with the restriction enzyme EcoRV and dephosphorylated using Fast AP (Thermo Fisher Scientific). Both linearized pcDNA3.1(+) fragments and amplified target gene fragments were purified using a DNA Gel Purification Kit (Tiangen) and quantified using a Nanodrop ND-2000 (Thermo Fisher Scientific). The pcDNA3.1(+) fragments and each target gene fragment were recombined using the MonClone Single Assembly Cloning Mix  and confirmed using DNA sequencing by Comate Bioscience Company Limited .The targets were amplified using Pfu  or Ex-taq  DNA Polymerase, with the prepared The endotoxin-free plasmids, pcDNA3.1(+)-alpha-2 and pcDNA3.1(+)-alpha-7.3, were prepared using the SanPrep Endotoxin-Free Plasmid Mini Kit (Sangon Biotech). The concentration was kept above 500 ng/μl to ensure that EDTA in the elution buffer did not interfere with the transfection assays. Primary mouse peritoneal macrophages were cultured in 6-well plates with RPMI 1640 complete medium  for 12 h, following which the cells were washed in warm PBS 3 times to remove penicillin and streptomycin and then cultured in 2 ml of RPMI supplemented with complete medium. The endotoxin-free plasmids, pcDNA3.1(+)-alpha-2 and pcDNA3.1(+)-alpha-7.3 (2.5 μg), were separately diluted in 125 μl of Opti-MEM reduced serum medium . Next, 5 µl of Lipofectamine 2000 transfection reagent  was diluted in 125 μl of Opti-MEM Reduced-Serum Medium. Liposome-DNA complexes were prepared by mixing the diluted endotoxin-free plasmid with Lipofectamine 2000 and allowing the mixture to stand at room temperature for 5 min. The complexes were separately transferred into the cells in each well and mixed slowly. After 4 h, the cell medium was replaced with 2 ml of RPMI 1640 complete medium and the culturing continued for a further 24 h. Fresh cell medium was added to the cells and cultured for different time points as per the assay design.Protein samples from supernatants and cell lysates were prepared as previously described . The mem2PO4, 187.5 mM NaCl, 25 mM HEPES and 125 mM NaHCO3. The mixture was centrifuged at 5000 g for 3 min, and the pellets were cross-linked with 10 µl DSS (25 mM in DMSO) and 40 µl ASC reaction buffer at 37 °C for 30 min. After centrifugation at 5000 g for 10 min, the pellets were dissolved in a solution of 40 µl ASC reaction buffer and 10 µl of 6× protein loading buffer , and the solution was then quenched at room temperature for 15 min, followed by boiling for 10 min. The protein samples were then subjected to western blot analysis using primary antibodies against ASC  at a dilution ratio of 1:500.Disuccinimidyl suberate (DSS) cross-linking was performed as previously described . The cel450nm values were converted to protein concentrations using an IL-1β standard curve.The cell culture supernatants were collected and used for the detection of the proinflammatory cytokine IL-1β secretions using the Mouse IL-1 Beta ELISA Kit , according to previously described procedures . The OD4Cells coated onto glass coverslips were gently washed 3 times in warm PBS, fixed in tissue cell fixative solution  for 10 min at room temperature (RT), permeabilized in 0.1% Triton X-100  at RT for 20 min and blocked in 5% bovine serum albumin (in PBS) at RT for 2 h. The cells were then separately incubated with primary antibody against ASC (1:100 dilution) or NLRP3 (1:100 dilution) at 4 °C overnight, and secondary antibody of Cy3-labeled goat anti-rabbit IgG(H+L)  or FITC-conjugated goat anti-mouse IgG  at 37 °C overnight for 1 h in the dark. Cell nuclei were stained with Hoechst 33258  for 5 min and observed under a fluorescence microscope .n = 7 per group): (i) PBS treatment negative control group ; (ii) MCC950 inhibitor [G. duodenalis cyst infection group ; and (iv) G. duodenalis cyst infection combined with group MCC950 inhibitor treatment group . The BW of each mouse was monitored daily, and all mice were euthanized on the 7th day. The harvested duodenum (3 cm long) was cut into small pieces in 1 ml of PBS, the cysts were disrupted in PBS at 4 °C overnight and the number of G. duodenalis trophozoites was counted under an optical microscope . Fresh duodenum (1 cm long) was isolated for hematoxylin and eosin (H&E) staining.Mice were divided into four groups (nhibitor  treatmenn = 7/treatment group): (i) the PBS treatment negative control group  injection  [G. duodenalis cyst infection positive control group (1.5 × 106 cysts/mouse by gavage); (iv) pcDNA3.1(+)-alpha-2 plasmid treatment group (100 µg/mouse DNA through i.m. injection); and (v) pcDNA3.1(+)-alpha-7.3 plasmid treatment group (100 µg/mouse DNA through i.m. injection). After 12 h, the mice in the MCC950 inhibitor group received MCC950 (10 mg/kg BW) i.p. daily for 7 days, whereas the mice in the MOCK group were treated with an equal volume of PBS. Blood samples were collected from the eyeballs of the mice and allowed to stand overnight at 4 °C. Serum samples were separated by enzyme-linked immunosorbent assay (ELISA) to measure the IL-1β levels.Mice were classified into two groups: (i) the MOCK control group and (ii) the MCC950 inhibitor group. In each group, there were five treatment methods ( muscle) , 29; (iin = 7/group). Group 1 was the PBS-treated negative control group: mice received 100 µl PBS through i.m. injection, followed by 100 µl PBS by gavage 3 days later. Group 2 was the G. duodenalis cyst infection-positive control group: mice were injected with 100 µL PBS and then gavaged with 1.5 × 106 cysts/mouse 3 days later. Group 3 was the pcDNA3.1(+) plasmid immunization combined with the G. duodenalis cyst infection control group: mice received 100 µg pcDNA3.1(+) plasmid DNA (i.m.) and were then gavaged with 1.5 × 106 cysts/mouse 3 days later. Groups 4 and 5 were the recombinant pcDNA3.1(+)-alpha-2 giardin plasmid or pcDNA3.1(+)-alpha-7.3 giardin plasmid immunization combined with the G. duodenalis cyst infection experiment groups: mice received 100 µg pcDNA3.1( +)-giardin plasmid DNA (i.m.) and were then gavaged with 1.5 × 106 cysts/mouse 3 days later. The BW of each mouse was monitored after G. duodenalis cyst gavage. Fresh duodenum was collected for parasite burden measurements and HE staining analysis.Thirty-five mice were divided into five groups  (P > 0.05).The in vitro and in vivo results were obtained from three technical replicates. Graphs were drawn using GraphPad Prism 7.00 . Differences between two groups were analyzed by the t-test, whereas differences among ≥ 3 groups were analyzed by one-way analysis of variance (ANOVA) using SPSS software . The homogeneity of variance of the data was analyzed using the Levene test, followed by a Bonferroni post-hoc test (B). Significance is indicated at Our previous Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in GEV proteomics showed that many targets may be involved in activation of the inflammasome signaling pathway . We seleF = 1.625, P = 0.1000; alpha-2 giardin: ANOVA, F = 1.625, P = 0.0007; alpha-7.3 giardin: ANOVA, F = 1.625, P < 0.0001; GEVs: ANOVA, F = 1.625, P = 0.0047) -alpha-2 giardin and pcDNA3.1(+)-alpha-7.3 giardin were separately transfected into primary mouse peritoneal macrophages, and the expression, location and oligomerization levels of key proteins of the NLRP3 inflammasome were determined. In this experiment, GEVs were used as the positive control group, and the no treatment group (PBS only) or pcDNA3.1(+) plasmid transfection treatment group were set as the negative groups. The results showed that, similar to the GEV treatment group, the recombinant plasmid DNA of pcDNA3.1(+)-alpha-2 giardin and pcDNA3.1(+)-alpha-7.3 giardin led to the upregulation of NLRP3, pro-IL-1β and pro-caspase-1 and the activation of caspase-1  MCC950 inhibitor treatment had no significant effect on the BW of mice. Compared with the single G. duodenalis cyst infection group, the BW in the MCC950-treated G. duodenalis cyst infection group was decreased at different levels  = 1.885, P = 0.0148; day 2: ANOVA, F = 0.4602, P < 0.0001; day 3: ANOVA, F = 0.8360, P = 0.0010; day 4: ANOVA, F = 1.683, P = 0.0052; day 5: ANOVA, F = 0.6497, P = 0.0645; day 6: ANOVA, F = 5.457, P = 0.0175; day 7: ANOVA, F = 2.893, P = 0.0202). These data suggest that the NLRP3 inflammasome was able to protect mice from significant weight loss in the early stage of G. duodenalis infection (2–4 days). Next, we aimed to detect G. duodenalis trophozoites in the duodenum lavage fluid, and the results are shown in Fig. G. duodenalis cyst infection group, the number of trophozoites in the duodenum was significantly increased following blocking of the NLRP3 inflammasome (t(12) = 2.902, P = 0.0133). Staining of the duodenum tissue with HE revealed that compared with the negative control group treated with PBS and MCC950 alone: (i) G. duodenalis cyst infection caused duodenal villi damage  = 0.4903, P = 0.0488) and crypt atrophy  = 0.4716, P = 0.0089); and (ii) the duodenum tissue of mice infected with G. duodenalis cysts and treated with the MCC950 inhibitor had no complete duodenum villi structure, and almost all duodenum villi were damaged and necrotic  = 0.4903, P = 0.0144) with crypt atrophy and branching  = 0.4716, P = 0.0481)  and NLRP3 inflammasome-blocked mice (MCC950 inhibitor treatment group), respectively. A detailed experimental scheme is shown in Fig. G. duodenalis cyst gavage treatment, the pcDNA3.1 intramuscular injection and the pcDNA3.1(+)-alpha-2 giardin or pcDNA3.1-alpha-7.3 giardin intramuscular injection treatments. Serum was collected on the seventh day after intramuscular injection of recombinant plasmids, and IL-1β levels in each group were determined. As shown in Fig. F = 4.062, P = 0.9998), whereas IL-β secretion levels were significantly upregulated in the G. duodenalis cyst infection group  = 4.062, P = 0.0002); (ii) pcDNA3.1-alpha-2 giardin and pcDNA3.1-alpha-7.3 giardin intramuscular injection significantly upregulated serum IL-1β levels  = 4.062, P < 0.0001); (iii) pcDNA3.1-alpha-7.3 giardin induced higher IL-1β secretion levels than those in the pcDNA3.1-alpha-2 giardin intramuscular injection group  = 4.062, P = 0.0333). In the MCC950 treatment group, compared with each group in the MOCK group: (i) the secretion levels of IL-1β in the PBS control group and pcDNA3.1 control group were downregulated to some extent after MCC950 inhibitor blocking, but the difference was not significant  = 3.540, P = 0.4912; pcDNA3.1: ANOVA, F = 3.540, P = 0.5949); (ii) after MCC950 blockade, IL-1β secretion was significantly downregulated in the G. duodenalis cyst infection group, pcDNA3.1-alpha-2 giardin group and pcDNA3.1-alpha-7.3 giardin group  = 3.540, P = 0.0120; pcDNA3.1-alpha-2 giardin: ANOVA, F = 3.540, P = 0.0447; pcDNA3.1-alpha-7.3 giardin: ANOVA, F = 3.540, P = 0.0164). These results suggest that alpha-2 giardin and alpha-7.3 giardin mediate the activation of the NLRP3 inflammasome in vivo.The secretion of IL-1β is a signature of inflammasome activation. To determine whether G. duodenalis, we used WT C57BL/6 mice and introduced alpha-2 giardin and alpha-7.3 giardin endotoxin-free plasmids by intramuscular injection, followed 3 days later by gavage with G. duodenalis cysts; the mice were then monitored for 7 days. A detailed experimental scheme is shown in Fig. G. duodenalis cysts, then it gradually increased. The activation of the NLRP3 inflammasome induced by intramuscular injection of alpha-2 giardin and alpha7.3 giardin significantly alleviated weight loss in mice  = 1.399, P = 0.9754; day 1: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 1.399, P = 0.9987; day 2: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.3172, P = 0.9979; day 2: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.3172, P = 0.8409; day 3: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.8222, P = 0.0262; day 3: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.8222, P = 0.0083; day 4: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.5620, P = 0.0012; day 4: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.5620, P < 0.0001; day 5: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.9728, P < 0.0001; day 5: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.9728, P < 0.0001; day 6: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.7154, P = 0.0012; day 6: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.7154, P = 0.0006; day 7: pcDNA3.1-alpha-2 giardin, ANOVA, F = 0.5369, P < 0.0001; day 7: pcDNA3.1-alpha-7.3 giardin, ANOVA, F = 0.5369, P < 0.0001). Parasite burden was evaluated in the duodenum  = 1.209, P = 0.0002; pcDNA3.1-alpha-7.3 giardin: ANOVA, F = 1.209, P < 0.0001). Moreover, the protective effect of alpha-7.3 giardin on mice was more significant than that of alpha-2 giardin  = 1.209, P = 0.0081). The HE staining results are shown in Fig. G. duodenalis-treated mice and mice injected with G. duodenalis combined with pcDNA3.1 empty vector, duodenal tissue lesions in mice injected with alpha-2 giardin and alpha-7.3 giardin were reduced, which was reflected by the increased villi damage  = 2.466, P = 0.0035 or P = 0.0068; pcDNA3.1-alpha-7.3 giardin: ANOVA, F = 2.466, P = 0.0028 or P = 0.0055) and reduced crypt atrophy  = 1.470, P = 0.0264 or P = 0.0158; pcDNA3.1-alpha-7.3 giardin: ANOVA, F = 1.470, P = 0.0371 or P = 0.0191). These results illustrate that alpha-2 giardin and alpha-7.3 giardin reduce the infection ability of G. duodenalis by activating the NLRP3 inflammasome in vivo.To investigate the effect of alpha-2 giardin- and alpha-7.3 giardin-mediated host NLRP3 inflammasome activation on the infection ability of Giardia duodenalis is a well-known intestinal parasite in humans and other mammals that causes giardiasis. In 2004, it was included in the WHO “Neglected Diseases Initiative” due to its high prevalence rates for 6 years, especially in communities of low socio-economic status [G. duodenalis infection. Murine macrophages have been reported to capture and kill G. duodenalis by releasing extracellular traps [G. duodenalis, a noninvasive extracellular parasite, activated the p38 MAPK, ERK, NF-κB p65 and NLRP3 inflammasome signaling pathways of murine macrophages to regulate the host inflammatory response and that this process was enhanced by released GEVs [c status . The innar traps . our presed GEVs , 24. HowStaphylococcus aureus [Escherichia coli [Bacillus cereus and non-hemolytic enterotoxin (NHE) from B. cereus [Toxoplasma gondii, Trichomonas vaginalis [Trypanosoma cruzi [Leishmania [Toxoplasma gondii virulence-related dense granule proteins GRA35, GRA42 and GRA43 are necessary for the induction of Lewis rat macrophage pyroptosis [Leishmania have focused on the single molecules that are involved in NLRP3 inflammasomes, such as parasite membrane lipophosphoglycan [G. duodenalis in a murine model [G. duodenalis virulence factors alpha-2 and alpha-7.3 giardins, which are unique to Giardia but relatively less reported. These two target genes were cloned into the eukaryotic expression system pcDNA3.1(+) vector and used for inflammasome activation determination.The NLRP3 inflammasome is located in the cytoplasm of immune cells and can be activated by various particles, such as uric acid crystals, toxins, bacteria, viruses and parasites. In studies on bacteria, toxins have been identified as key PAMPs that activate inflammasome sensors, leading to inflammation and cell death . Some sts aureus  and Eschhia coli , hemolys. cereus , have be. cereus  and Zika. cereus , are vitaginalis , Trypanoma cruzi  and Leisishmania . Toxoplahoglycan  or zinc-hoglycan . Among tne model . In our G. duodenalis infection study. The results showed that many alpha-giardins are responsible for canonical inflammasome activation, which is consistent with the findings of key virulent molecules involved in bacteria and viruses. However, our study was just a preliminary screening, and there are also other molecules that may activate non-canonical inflammasomes, as both canonical and non-canonical inflammasomes have been found to exist in G. duodenalis infection, as reported in our previous study [G. muris or E. coli EPEC strain alone could upregulate the fluorescence intensity of NLRP3, ASC and caspase-1, although not significantly, while co-stimulation with G. muris and E. coli increased the levels of the three proteins [ Giardia species, cell lines and primary cells. We also carried out in vivo assays using MCC950 in 5-week-old female WT C57BL/6 mice, which are more susceptible to G. duodenalis. MCC950 is a potent and selective small-molecule inhibitor of NLRP3 that blocks both classical and nonclassical NLRP3 activation at nanomolar concentrations. MCC950 inhibits NLRP3 activation but does not influence the activation of AIM2, NLRC4 and NLRP1 inflammasomes or the TLR signaling pathways [+ outflow, Ca2+ influx or the interaction between NLRP3 and ASC; instead, it inhibits NLRP3 inflammasome activation by blocking ASC oligomerization [G. duodenalis alpha-2 giardin and alpha-7.3 giardin were able to activate murine NLRP3 inflammasomes.In our mouse model, cleaved caspase fragments acted as the markers of inflammasome activation. Once stimulation occurs, NLRP3 interacts with ASC, recruits pro-caspase and generates active caspase, which cleaves pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18, respectively. Inflammatory caspases , a family of conserved cysteine proteases, are critical for innate defenses and are involved in inflammation and programmed cell death . Caspaseus study . To furtproteins . This dipathways . MCC950 rization . TherefoG. muris, inducing IL-17RA signaling, generating antimicrobial peptides and regulating complement activation [Giardia infection is more likely to occur in young individuals, and it has been reported that Giardia muris infection in young mice could not activate the IL-17A response to function in its protective roles [Giardia infection. The authors of a recent study reported that G. muris could promote the production of antimicrobial peptides and reduce its attachment capacity and the number of trophozoites in the intestinal tract by activating the NLRP3 inflammasome with the aid of E. coli EPEC, thus weakening the severity of the E. coli-induced disease [Pseudomonas aeruginosa triggers macrophage autophagy to escape cell death and that this process is dependent on the activation of the NLRP3 inflammasome [ Neospora caninum, reactive oxygen species-mediated NLRP3 inflammasome activation restricts its replication in hosts, making it a potential therapeutic target [Paracoccidioides brasiliensis was found to induce NLRP3 inflammasome activation in mouse bone marrow-derived dendritic cells, leading to the release of the inflammatory cytokine IL-1β, which plays a vital role in host defense [Leishmania spp., including L. amazonensis, L. major, L. braziliensis and L. infantum chagasi, activate NLRP3- and ASC-dependent caspase-1 in macrophage cells, and the infection of Leishmania spp. in NLRP3/ASC/caspase-1 gene-deficient mice intensified parasite replication [Leishmania infection triggered the activation of the NLRP3 inflammasome in macrophages, which limited intracellular parasite replication. Therefore, Leishmania may inhibit NLRP3 activation as an avoidance strategy. In in vivo studies, the NLRP3 inflammasome promoted Leishmania clearance, but not in tissues [Shigella bacteria is one of the major bacteria responsible for diarrhea worldwide. These bacteria can induce IL-1β production through P2X7 receptor-mediated K+ outflow, reactive oxygen species, lysosomal acidification and mitochondrial damage. The NLRP3 inflammasome negatively regulates phagocytosis and the bactericidal activity of macrophages against Shigella [Plasmodium demonstrated that AIM2-, NLRP3- or caspase-1-deficient mice infected with Plasmodium produced high levels of type-1 interferons and were more resistant to Plasmodium infection [Ample evidence has accumulated in the last decade showing that IL-17A is the central regulator of immunity against tivation . Howeverve roles , which l disease . The NLRammasome . For Neoc target . Paracoc defense . Severallication . Zamboni tissues . Convers tissues . ShigellShigella . Studiesnfection . HoweverG. duodenalis can activate the host NLRP3 inflammasome through alpha-2 giardin and alpha-7.3 giardin and reduce the pathogenicity of G. duodenalis in mice.  In the present study, inhibition of the NLRP3 inflammasome with MCC950 reduced the BW of mice and increased the number of trophozoites in the intestinal lavage fluid, resulting in more severe pathological changes in duodenal tissues. Alpha-2 giardin and alpha-7.3 giardin activated the host murine NLRP3 inflammasome, increased mouse BW, decreased the amount of trophozoites in intestinal lavage fluid and attenuated pathological duodenal lesions. These results suggest that G. duodenalis infection ability in mice. As such, these molecules are promising targets for the prevention of giardiasis.In summary, our result show that alpha-2 and alpha-7.3 giardins triggered activation of the host NLRP3 inflammasome and decreased


Human activities can significantly impact the natural ecosystem. As an important part of terrestrial ecosystems, soil participates in energy and material cycle. With the continuous intensification of human activities, soil undergoes increasingly serious disturbance. Under the influence of global change and human activities, the variability of heavy metals in soil is worthy of further discussion. Taking Lujiaowan and Juhuatai Scenic Spot on the northern slope of Tianshan Mountain as the research area, this study investigated the concentrations of Pb, As, Zn, Cu and Mn in the soil and analyzed their distribution and pollution levels. Meanwhile, the main sources of soil heavy metals were explored using autocorrelation analysis and principal component analysis. Results showed that the order of the average concentration of heavy metals in the study area was as follows: Mn> Zn> Cu> Pb> As. None of them exceeded the national soil environmental quality level II standard, and the average concentrations of Zn, Cu, and As exceeded the background value of Xinjiang soil, reaching a light pollution level. In addition, the distribution of heavy metals in soil displayed a regular trend, and a positive correlation was found between disturbance intensity and heavy metal concentration. The geoaccumulation index also showed that the five heavy metals in the study area had lower pollution degree compared with the background value in Xinjiang. The order of potential ecological risk was As > Cu > Pb > Zn > Mn. As an important natural resource, soil plays an important role in absorbing and purifying pollutants in the environment. Soil pollution occurs when the pollutant concentration in soil exceeds a specific value, consequently influencing soil structure, function, and plant growth –3. EcosyExisting research shows that tourism activities significantly affect the concentration of heavy metals in the soil near scenic spots, and most studies believe that tourism activities increase the concentrations of some heavy metals in soil , 17, 18.Actually, the concentration of soil heavy metal might be correlated to the intensity of human disturbances, which indicates human pressure on the landscape . CompareTaking the northern slope of Tianshan Mountain as the research object, the present study selected two typical tourist areas (Lujiaowan and Juhuatai) to analyze the distribution and pollution degree of soil heavy metals under different tourism disturbance intensities and to explore the main sources of soil heavy metals. Combined with the geoaccumulation index method and potential ecological risk index method, the potential ecological risk level of soil heavy metals in the study area was evaluated. The corresponding results could provide reference for the environmental protection of the arid regions in Central Asia.Lujiaowan scenic area is located between 84°56′–85°26′E and 43°49′–44°04′N, with an altitude of more than 4000 m. The average temperature in summer is 20°C, the average temperature in winter is -14°C, and the annual rainfall is more than 600 mm. Juhuatai scenic spot is located at 87°10′–87°15′E, 43°29′–43°47′N, with an altitude of 2000–2400m, an average annual temperature of 2°C–4°C, a frost-free period of 130–150 days, and an annual precipitation of 270–300 mm. The vegetation of these two spots mainly includes coniferous forest and grassland. The soil is mainly composed of Mountain grey cinnamon soil.Soil samples were collected in May 2019. Sampling information is presented in Tables Pi is the single factor index of metal i in soil, then Pi= Ci/Si, where Ci represents the measured concentration of metal i in soil and Si is geochemical background value as mentioned previously. According to the value of Pi, the environmental pollution could be categories to four classes as shows in The pollution index (single factor) is widely used in evaluating the heavy metal pollution in various environments –26. If dIgeo) was used to evaluate the heavy metal pollution in our study area. The Igeo was defined by Müller [Igeo= log2(Cn/1.5 × Bn), where Cn is the concentration of heavy metals in soil and Bn is the geochemical background value. According to Müller [Igeo  to class 5 .In the present study, the geoaccumulation index  has been widely used since it was introduced . The RI Since it was proved that two heavy metals with a significant correlation coefficient may share the same origin, then correlation analysis was used to identify the origin of elements. PCA is a statistical method that transforms multiple variables into a few principal components. It is an important dimension reduction method that can reduce the dimension of high-dimensional data and the number of predictive variables , 30. In The horizontal distribution of soil heavy metal concentration is shown in At Juhuatai, the maximum concentrations of soil Pb, As, and Mn appeared at 2.5 m away from sampling centers and decreased with increasing distance . Among t-1, 11.18 ± 2.84 mg·kg-1, 77.53 ± 11.08 mg·kg-1, 31.93 ± 7.87 mg·kg-1 and 560.93 ± 88.72 mg·kg-1, respectively. In 10–20 cm depth, the average concentration of Pb, As, Zn, Cu, Mn are 14.78 ± 4.43 mg·kg-1, 11.05 ± 2.37 mg·kg-1, 72.57 ± 9.61 mg·kg-1, 35.28 ± 8.55 mg·kg-1 and 573.30 ± 87.94 mg·kg-1, respectively. At Juhuatai, the average concentration of Pb, As, Zn, Cu, Mn in 0–10cm soil are 17.32 ± 3.83 mg·kg-1, 11.08 ± 2.69 mg·kg-1, 84.87 ± 18.27 mg·kg-1, 27.84 ± 6.03 mg·kg-1 and 546.52 ± 121.46 mg·kg-1, respectively. In 10–20 cm depth, the average concentrations are 16.81 ± 2.72 mg·kg-1, 11.58 ± 2.38 mg·kg-1, 82.25 ± 21.04 mg·kg-1, 24.75 ± 4.29 mg·kg-1 and 523.98 ± 99.30 mg·kg-1, respectively. It can be noted that not only the average concentrations of heavy metal in 10–20 cm soil are relatively lower, the variations (as denoted by standard deviation) of concentrations are also lower in the 10–20 cm soil, compare with 0–10 cm soils. The relatively low variation of concentration in deeper soil are in line with previous studies [The average concentration of Pb, As, Zn, Cu, Mn in 0–10cm soil of Lujiaowan are 15.73 ± 3.02 mg·kg studies –38, sincIn the 0–10 and 10–20 cm depth soils of Lujiaowan, the Pi values of Zn were 1.30 and 1.03 in the soil of the no-disturbance area, 1.04 and 1.03 under moderate disturbance, 1.14 and 1.10 under severe disturbance, and 1.15 and 1.04 in the soil near the hiking trail, respectively . The conIn the 0–10 and 10–20 cm soil layers of Juhuatai scenic spot, the Pi values of As were 0.85 and 1.13 in the soil of no disturbance, 1.07 and 1.11 under severe disturbance, and 1.03 and 1.09 near the hiking trail, respectively . The con-1, 11.37 mg·kg-1), Zn  and Cu  were higher than the background values of Xinjiang soil, reaching the level of light pollution. Generally, the concentrations of heavy metal were higher in moderate disturbance, severe disturbance and hiking trial area compare with no disturbance area, as shows in The order of average concentration of heavy metals in the soil of the study area was Mn > Zn > Cu > Pb > As . In the The correlation coefficients between Pb and As and between Mn and As were 0.79 and -0.77 (p < 0.05), respectively . Therefo-1 to 58.06 mg·kg-1, with a median of 6.0 mg·kg-1 [The PCA revealed that the contributions of the first two principal components were 59.12% and 26.40%, respectively, and the cumulative variance was 85.52% at Lujiaowan . Among t mg·kg-1 . The ant mg·kg-1 . Therefo mg·kg-1 .geo of the five heavy metals in soil under different disturbance conditions in the study area was less than 0 , Cu , and As  in the soil of Lujiaowan and Juhuatai scenic spots were higher than the background values of soils in Xinjiang. To the extent of light pollution.The concentrations of Pb, As, Zn, Cu and Mn in soils of our study area were lower than the national II standard for soil environmental quality. In the 0-10cm soil layer, the average concentrations of Zn Click here for additional data file.


Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA. FXN gene that leads to gene silencing and decreased levels of the mitochondrial protein frataxin (Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder with an incidence of 1 in 29,000 (1). Currently it has no approved treatment . FRDA isfrataxin . The numfrataxin , 6. The 2 and NADH that can be utilized to maintain the electrochemical gradient across the inner mitochondrial membrane needed for ATP synthesis. Therefore, increased lipid metabolism observed in FRDA could be important to maintain cellular homeostasis during mitochondrial dysfunction.One function of frataxin is in the formation of the iron-sulfur (Fe-S) cluster prosthetic groups that are critical for enzymes in the Krebs cycle (aconitase), oxidative phosphorylation (electron transport chain components of complexes I–III), and fatty acid breakdown (β-oxidation) , 8. FratA recent study found reactive oxygen species-independent accumulation of iron in the nervous system of an FRDA fly model with a mutant frataxin homolog, associated with enhanced sphingolipid synthesis . SphingoCeramides are central intermediates in sphingolipid metabolism and have been implicated in several cellular processes including apoptosis . DysreguIdeally, metabolic and lipidomic abnormalities should be studied in the most affected tissues, but frataxin deficiency is present in all tissues to different extents . Since iNine patients with FRDA and nine healthy controls were entered into the study with no significant differences between the two groups in terms of mean ± SD sampling ages  . There wDMEM low glucose was purchased from Life Technologies . HyClone fetal bovine serum (FBS), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), isopropanol, and Optima-grade water, methanol (MeOH), methyl-tert-butyl ether and acetonitrile (ACN) were purchased from Thermo Fisher Scientific . As detailed in 6 cells, after incubation with fresh media for 12 h, so all cells would be at the same feeding stage. One of the plates was used for cell counting and all the numbers were within 10% of 1∗E6 per plate. For stable isotope tracing experiment, overnight seeded fibroblasts had their media change with fresh medium containing 100 μM 13C16-palmitate for 6 h. 12C-palmitate medium, 100 μM, was used as control for background subtraction. The medium was aspirated, and cells were washed two times with 5 ml 0.9% NaCl before freezing at −80°C.Primary fibroblasts from the skin biopsy samples were isolated and established according to detailed procedures as previously published , 21. Theet al.  5-sulfosalicylic acid in water. Three microliter injections were used for LC-HRMS analysis with metabolomics method.Metabolomic extraction protocol for fibroblasts was adapted from Guo et al. . A volumet al. . Cells wLipid extraction protocol for fibroblasts was adapted and modified from previous publication . BrieflyoC) and column heater (50oC). Solvent A was 5 mM DIPEA and 200 mM HFIP and solvent B was 5 mM DIPEA and 200 mM HFIP in methanol. Flow gradient conditions were as follows: 0% B for 2 min at 0.18 ml min−1, increased to 1% B for 2 min at 0.2 ml min−1, increased to 2% B for 4 min, increased to 14% B for 2 min, increased to 70% B for 2 min, increased to 99% B for 1 min, increased flow rate to 0.3 ml min−1 for 0.5 min, increased flow rate to 0.4 ml min−1 for 4 min, then washed by decreasing to 0% B for 2.3 min at 0.3 ml min−1, decreased to 0.2 ml min−1 for 0.2 min, and ending with flow of 0.18 ml min−1. Samples were analyzed using a Q Exactive HF (QE-HF)  equipped with a heated electrospray ionization source operated in the negative ion mode. Source parameters were the same as described before   on a Dionex UltiMate 3000 quaternary UHPLC  equipped with a refrigerated autosampler (5d before . Data ac°C), and the column heater was set to 35°C. Solvent A was 1/1 ACN/water with 10 mM ammonium formate and 0.1% formic acid. Solvent B was of 10/88/2 ACN/isopropanol/water with 2 mM ammonium formate and 0.02% formic acid. Flow rate was 0.4 ml min−1. Flow gradient conditions were as follows: 0 min, 90% A; 1 min, 90% A; 4 min, 60% A; 12 min, 25% A; 21 min, 1% A; 24 min, 1% A; 24.1 min, 90% A; 28 min, 90% A. Samples were analyzed using the QE-HF operated in positive ion mode, then in negative ion mode. Column effluent was diverted to the QE-HF from 2 to 23 min and then to waste for the remaining time of the run.Lipids were separated using an Accucore C18 HPLC column   on an UltiMate 3000 quaternary UHPLC equipped with a refrigerated autosampler  @ 15,000 were generated with AGC target = 1e5, Maximum IT=25 ms, and (N)CE/stepped nce = 25, 30, 35v. For targeted analysis of ceramide isotopomers, data acquisition was performed in Full Scan/DIA mode. The Full Scan settings is the same with the above setting, for DIA setting, an inclusion list with ceramide was included with scheduled scan window 1 min; other settings: resolution 15,000, AGC target=1e5, maximum IT=auto, isolation window = 1.0 m/z, loop count=30.Data acquisition was performed in Full Scan/ddMS2 mode @ 120,000 resolution. The Full Scan settings were as follows: AGC target, 1e6; Maximum IT, 250 ms; scan range, 250–1800 t test. Statistical analysis was conducted using GraphPad Prism v9.0 for Windows.For metabolomics data analysis, quantification of chromatographic peaks was performed with Xcalibur 3.0 (Thermo) from full scan data. The approximate nanogram per plate for each compound was calculated by multiplying the area ratio , but no significant difference in CoQ10 level was observed between FRDA and control fibroblasts  profiling in the same assay, and the levels of several acyl-CoA molecules, including CoASH, acetyl-CoA, succinyl-CoA, butyryl-CoA, isobutyryl-CoA, malonyl-CoA, HMG-CoA, β-hydroxybutyrate (BHB)-CoA, and propionyl-CoA F, were qroblasts I. Levelsroblasts . In agreroblasts B, but BHP value using t test and fold changes (FCs) that were defined as the ratio of FRDA to control average response measured by the signal intensity for each ion. Significantly changed metabolites (SCMs) were filtered by P value less than 0.05 and FC higher than 1.5 (P less than 0.05)  , ceramides (Cers), cholesterol esters, diglycerides, hexosyl-ceramides, lysophosphatidylcholines, lysophosphatidylethanolamines, lysophosphatidylinositols, phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidylinositols, phosphatidylserines (PSs), sphingomyelins (SM), and triglycerides (TGs). PCA was used to compare the similarities of the lipidomic profiles between FRDA and control fibroblasts. The score plot of the PCA model showed that the FRDA and control fibroblasts are clearly separated on the PC2 A, implyithan 1.5 C, which than 1.5 D. Ceramithan 1.5 D. Severathan 1.5 , we perfthan 1.5 E. A totathan 1.5 F, includhan 0.1) .Fig. 2FRP=0.028)   B. d18:0-han 0.05 C. d18:1-han 0.05 B, with mhan 0.05 C. d18:2-han 0.05 . d18:2-CP=0.012) B. LevelsP=0.012) , and SM It is worth mentioning that we could not identify in the fibroblast cells Cer (18:1/20:4) which was one of the most increased in the heart tissues from patients with FRDA reported by the Bellen group.  We did nP=0.066). d18:0-Cers (P=0.014) and d18:1-Cers (P=0.05) were more strongly correlated with the GAA1 repeat length than d18:2-Cers (P=0.251). Individual ceramides belonging to d18:0-Cers and d18:1-Cers showed strong positive correlation with GAA repeat length with r > 0.7 and P < 0.05 (P=0.054) and d18:1-Cers (P=0.053) negatively correlated with the frataxin levels, and most of the classical ceramides (d18:1-Cer) had negative correlations with the frataxin levels with P<0.1 or around 0.05  and could be mapped to the “Sphingolipid metabolism” (KEGG pathway map hsa 00600)  .13C16]-palmitate in several fibroblast lines. A targeted-HRMS strategy-parallel reaction monitoring was used for the analysis of mass isotopomer distribution of ceramide or Hex1Cer . This method allowed us to sensitively and selectively detect ceramide or Hex1Cer with no-chain-labeled, fatty-acid-chain-labeled, sphingosine-base-labeled, and dual-labeled forms simultaneously  and Cer(d18:1/16:0) were more similarly increased in all three tested FRDA lines, but the Cer(d18:2/16:0) was differently increased between cell lines. We also observed higher isotopic enrichment for very-long-chain ceramides, such as Cer(d18:1/24:0) and Cer(d18:1/24:1) B, which P=0.076) (P=0.001) , AcCa (18:2), AcCa (20:1), AcCa (20:3), AcCa (20:4), AcCa (22:4), and AcCa (24:1), were increased in FRDA fibroblasts A, implyiP=0.076) E, and a P=0.001) F. SimilaP=0.001) G–I. InteThe enrichment of PUFA in lipids was also observed in TGs composition A. PUFA-TSimilarly with the levels of TGs, the PGs levels were significantly increased in FRDA fibroblasts J; most oThe data from the fibroblast cells showed that the dysregulation in lipid metabolism extends in patients with FRDA to unaffected tissues (fibroblasts). Our results regarding increased ceramides in FRDA fibroblast cells replicate the findings from the heart tissue  of patieCeramides are one of the most studied classes of bioactive sphingolipids, as they regulate stress resistance, proliferation, differentiation, and maturation of different cells . Local oCeramides are believed to be a main factor in reorganizing microscopic membrane rafts into signaling platforms in response to stress.  CeramideAcylcarnitines are important lipid biomarkers reflecting acyl-CoA status  and fattApart from having the role as biomarkers, acylcarnitines also possess bioactive and inflammatory properties and can regulate the function of membrane-based systems and impact cell biology.  In cardiAs mentioned, the metabolites BHB-CoA and HMG-CoA accumulated in FRDA fibroblast cells G, but acHMG-CoA serves as the precursor to isoprenoid groups that are incorporated into a wide variety of end-products, including cholesterol, ubiquinone, heme, and dolichol. Our lipidomic profiling showed that two important products of HMG-CoA, CoQ10 and cholesterol, are not affected by frataxin deficiency, which may explain why improvements by idebenone  were not statistically significant in clinical trials . SeveralNotably, PUFAs were enriched in phospholipids and triglycerides (both elevated in FRDA fibroblasts) , which wAltogether, our work demonstrates that the levels of ceramides and acylcarnitines, directly correlate with disease severity in FRDA, making them possible biomarkers of disease, as accurate assays to quantify small molecules and lipids are less labor intensive than assays for accurate protein quantification. Furthermore, the isotope tracing experiment suggested the mechanism for ceramide accumulation, from de novo biosynthesis rather than complex lipid turnover. Currently there are several US Food and Drug Administration-approved drugs targeting ceramides synthesis that are extensively detailed by Kovilakath and Coward . Togethemesaros@upenn.edu.All data are contained within the article or supplemental material. Raw mass spec file data will be shared upon request from the corresponding author, Clementina Mesaros, PhD, Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, e-mail: The Supporting Information is available free of charge.This article contains The authors declare that they have no conflict of interest with the contents of this article.


The resulting composite material (Tb-COF) was fully characterized by geometric analysis, IR, XRD, porosity analysis, SEM/TEM, and elemental analysis. It was found that Tb(DPA)33- was loaded into the hexagonal cage in an I-COF host with an ionic exchange ratio of 41%. The as-synthesized Tb-COF showed weak Tb(III) emission and strong red COF emission, after adding PA, Tb(III) emission was increased whereas COF emission weakened greatly, showing sensing behavior. Linear working curves were observed with good selectivity. The sensing mechanism was revealed as follows. PA molecules replaced the [Tb(PDA)3]3- component trapped in Tb-COF, releasing free luminescent [Tb(PDA)3]3-. After incorporating PA in the hexagonal cage, the COF emission was quenched. This sensing mechanism ensured a good selectivity over competing species, including cations, anions, and nitrocompounds. The adsorption and removal performance of I-COF for PA were investigated as well.Picric acid (PA) is an important chemical product which has been widely used in dye manufacturing, antiseptics, and pharmaceuticals. Owing to PA’s extreme electron-deficient structure, its natural degradation is hard, leading to accumulation in the environment and finally threatening the ecosystem and human health. In this case, PA detection and removal becomes more and more important, concerning environmental protection and human health. In this study, an ionic covalent organic framework (I-COF) was synthesized and modified with a luminescent Tb(III) emitter (Tb(DPA) It seems that the recognition and removal of PA may be carried out by a one-step procedure using a proper optical sensing platform, where its sensing probe gives a sensing signal for PA and its supporting host adsorbs PA.Picric acid (PA) is an important chemical product which has been widely used in dye manufacturing, antiseptics, and pharmaceuticals . Owing tAs for the sensing probe, PA requires a stable emitter since its electron-deficient structure tends to quench most emitters. It appears that rare earth-based probes are promising ones for PA detection. These metal-centered emitters have long-lived sharp emission lines. Chemical modifications and reactions exert a slim effect on these emission lines, making them a promising sensing probe for PA detection. As for the supporting host, porous materials with stable structure and high surface area-to-volume ratio are desired . Ever siThere are, however, two issues when changing these organic building blocks. First, the structural change of resulting COF is sometimes unpredictable when changing organic building blocks, since the basic geometry of the core framework may be changed as well. Second, the organic building blocks of COFs are usually neutral ones, so that the resulting COFs are crystalline polymers with no charge, which limits the further modification and grafting of functional groups. For example, in the field of optical sensing, sensing probe should be immobilized and dispersed in supporting host to guarantee fluent dispersal and diffusion of analyte molecules. Although COF materials satisfy most requirements for an ideal supporting host, such as uniform pore size distribution, enough mechanical strength, stable, and versatile structure, there is an important issue for COFs, which is their neutral structure.via van der Waals’ force or covalently grafted to the organic backbone of COFs 33- was selected as the sensing probe for ionic exchange. The combination of the ionic COF and Tb(DPA)33- is supposed to realize optical sensing signals for picric acid.Mandal S. and coworkers have reported and demonstrated a series of well-designed MOF-based materials for the selective sensing/removal of phosphate ions, Hg(II) ions, Fe(III) ions, nitro aromatic explosives, and practical applications . These azeolites . As show3Tb(DPA)3 as starting compounds. All chemicals used in this work were AR grade ones purchased from Aldrich Chemical Company, including dioxane, mesitylene, 3,8-diamino-5-ethyl-6-phenylphenanthridin-5-ium bromide, picric acid standard sample, pyridine-2,6-dicarboxylic acid (H2DPA), NaOH, Tb(Cl3)•nH2O, phloroglucinol, hexamethylenetetramine, trifluoroacetic acid, and p-toluenesulfonic acid. Organic solvents were purified with standard protocols.The synthetic route and sensing strategy of I-COF and Tb-COF is shown in 2 adsorption and desorption experiment was performed on a Quantachrome Autosorb iQ2 analyzer at 77 K (liquid nitrogen). The 13C NMR spectrum was recorded using an Avance III 400 WB spectrometer , using adamantine as reference. 1H spectra were recorded on this spectrometer with tetramethylsilane as reference (300 MHz). Thermal analysis was performed on a NETZSCH STA 449C thermal analyzer (10°C/min). Elemental analysis was performed by a Carlo Erba 1106 elemental analyzer. XPS (photoelectron spectroscopy) spectra were analyzed on an ESCALAB 250 (Thermo Scientific) spectrometer using Al Ka radiation (1486.6 eV). ICP data were collected by a Shimadzu ICPs-1500D spectrometer. Thermogravimetric analysis (TGA) was performed on a Perkin–Elmer thermal analyzer.Powder X-ray diffraction (PXRD) was performed on a D/MAX2550 (Riguku) diffractometer using Cu-K radiation at 50 kV and 200 mA. IR spectra were recorded by using a Fourier transform IR spectrometer (IFS 66V/S). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were taken from an S-4800 microscope (Hitachi) and a JEM-2010 microscope (JEOL), respectively. UV–Vis and emission spectra were recorded using a UV-3101 PC spectrometer (Shimadzu) and a F7000 spectrometer (Hitachi), respectively. The N3[Tb(DPA)3] was synthesized as follows: TbCl3 6H2O (2 mmol) and DPA (6 mmol) were mixed in H2O (20 ml). NaOH solution (1 M) was added to adjust the pH to 5.2. After vaporizing solvent water under reduced pressure (100 kPa), the solid product was collected and recrystallized in water. Anal. Calcd. For C21H37N3O26Na3Tb (Na3Tb(PDA)3 14H2O): C, 25.86; H, 3.82; and N, 4.31. Found: C, 25.70; H, 3.97; and N, 4.12. Its single crystal structure was obtained and discussed later to confirm its identity.The sensing probe Na2 stream, then heated at 100°C for 150 h. After cooling, the organic phase was extracted with CH2Cl2 (100 ml x3) and vaporized to give pale yellow solid. Yield: 11%. Anal. Calcd. For C9H9O6: C, 51.44; H, 2.88; N, 0.0. Found: C, 51.29; H, 2.93; N, 0.05 13C NMR (CDCl3) δ 196.2, 172.6, and 103.2.I-COF was synthesized using 3,8-diamino-5-ethyl-6-phenylphenanthridin-5-ium bromide and 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde as starting compounds. 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde was first synthesized as follows: a mixture of hexamethylenetetramine (108 mmol), phloroglucinol (48 mmol), and trifluoroacetic acid (90 ml) was stirred under N°C for 12 h to get I-COF. Yield: 72%. Anal. Calcd. For C162H120N18O12Br6: C, 65.07; H, 4.04; and N, 8.43. Found: C, 65.25; H, 4.17; and N, 8.49. 13C NMR (solid) δ 190.5, 167.1, 150.3, 144.7, 135.9, 130.4, 127.8, 113.6, 47.3, and 18.4.The above obtained 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde (2 mmol) was mixed with 3,8-diamino-5-ethyl-6-phenylphenanthridin-5-ium bromide (3 mmol), and then dissolved in a mixed solvent of dioxane (10 ml), mesitylene (10 ml) and aqueous acetic acid . This solution was degased and sealed in a Pyrex tube. After being heated at 120°C for 3 days, red-purple solid product was collected, dispersed in THF/ethanol, and stirred for 24 h. The final product was dried at 100via anionic exchange with Na3[Tb(DPA)3] as follows: I-COF (1.5 g) was dispersed in 20 ml of ethanol/H2O (v:v = 1:1), then excess Na3[Tb(DPA)3] (3.0 g) was added. This mixture was stirred at room temperature for 24 h and then filtered off. Such anionic exchange procedure was repeated for another four times. The final product was flushed with water and dried in vacuum at 120°C for 2 days to give Tb-COF. Yield: 85%. Anal. Calcd. For C162H120N18O12Br3 C21H9N3O12Tb1 (assuming that 50% of Br− ions were replaced by [Tb(DPA)3]3-: C, 64.56; H, 3.82; and N, 8.61. Found: C, 64.61; H, 3.90; and N, 8.55. 13C NMR (solid) δ 190.6, 169.2, 167.4, 160.0, 150.7, 145.1, 136.4, 134.9, 131.1, 126.5, 114.7, 46.9, and 19.1.Tb-COF was synthesized 2O . The resulting mixture was treated in ultrasonic bath for 5 min. Then, PA standard was finely weighted and mixed with Tb-COF solution. Before sending to the spectral experiment, each sample was treated in ultrasonic bath for 3 min. For the PA adsorption experiment, Tb-COF solid was finely weighted and mixed with PA standard solution . The mixture was treated in ultrasonic bath and then centrifuged  for 10 minutes before sending to quantification.For sensing performance evaluation, Tb-COF standard suspension was first prepared by dispersing 0.4 g of Tb-COF in a mixed solvent of ethanol:H3Tb(PDA)3 single crystal structure was obtained (CCDC 1185465). Some of its key structural parameters are listed in 3Tb(PDA)3 crystal, with slight distortion. There are four [Tb(PDA)3]3- components in a cell unit, so that the cell length values (b and c) are as large as ∼18 Å, but the cell length of a is only 9.6 Å. As shown in + ions) are randomly distributed outside of the [Tb(PDA)3]3- coordination sphere to meet the charge balance. The maximum diameter of [Tb(PDA)3]3- is measured as ∼11.4 Å using a Na3Tb(PDA)3 crystal. The small size of [Tb(PDA)3]3- component makes it convenient to be loaded in the COF matrix, which will be confirmed later.The Na3]3- component. There are three Br− anions in each hexagonal cage, which allows the ionic exchange of one [Tb(PDA)3]3- component to meet the charge balance after ionic exchange. There are three possible alignment modes for I-COF layers, which are ideal AA stacking, ideal staggered AB stacking, and offset ABA stacking. The geometrical energy minimization by the universal force field Forcite module suggests that the offset ABA stacking has the lowest total energy  compared to those of ideal AA stacking  and ideal staggered AB stacking . In this case, it is assumed that the offset ABA-stacking mode is applied by I-COF solid which is energetically favored.3Tb(PDA)3, indicating that the I-COF matrix has been well preserved after ionic exchange.This statement is strengthened by the XRD comparison between I-COF solid and the aforementioned three stacking modes. It is observed from 3Tb(PDA)3 are shown in 3Tb(PDA)3, the broad IR bands around 3,523 cm−1 and 1,603 cm−1 are attributed to C=O vibration and stretching. As for I-COF, the strong IR bands at 1,590 cm−1 and 1,268 cm−1 are named as vibrations from C=C and C-N bonds. These characteristic bands are all found from the IR spectrum of Tb-COF. There are no new IR bands, indicating that Na3Tb(PDA)3 has been well-preserved in Tb-COF.The IR spectra of I-COF, Tb-COF, and Na3Tb(PDA)3 in I-COF is first confirmed by porosity comparison between I-COF and Tb-COF. As shown in 2 uptake is observed for I-COF at a low pressure of 0–0.1 P/P0, which reveals its microporous network. The Brunauer–Emmett–Teller (BET) surface area of I-COF is measured as 770 m2/g, with a pore size of ∼17.0 Å. This value is smaller than the diameter of the simulated I-COF monolayer hexagonal cage (22.3 Å), but still larger than the maximum diameter of [Tb(PDA)3]3- (∼11.4 Å). In this case, it is assumed that, theoretically, there will be only one [Tb(PDA)3]3- component in each I-COF hexagonal cage, to meet room requirement and charge balance. After loading procedure, [Tb(PDA)3]3- component occupies I-COF hexagonal cages, resulting in the rather small BET surface of Tb-COF (<10 m2/g).The successful loading of Na3]3- component has been incorporated into the hexagonal cage of I-COF, forming Tb-COF. No obvious signal from Na indicates the successful ionic exchange. On the other hand, Br element is still observed in Tb-COF, indicating an incomplete ionic exchange between the Br− and [Tb(PDA)3]3- components.The SEM/TEM images of Tb-COF, along with its elemental mapping and EDX data, are shown in 162H120N18O12∙2[C21H9N3O12Tb1], with an elemental analysis of C, 64.15; H, 3.64; and N, 8.80. As aforementioned, I-COF allows only one [Tb(PDA)3]3- component in each hexagonal cage, which is 50% ionic exchange, and its molecular composition should be C162H120N18O12Br3∙C21H9N3O12Tb1, with an elemental analysis of C, 64.56; H, 3.82; and N, 8.61. Clearly, the experimental data support a 50% of ionic exchange. For a more direct evaluation on the doping level, the Br and Tb contents of Tb-COF are determined as 9.8 and 3.2 ppm, respectively, corresponding to a molar ratio of 6.08:1 and suggesting an ionic exchange ratio of 33% . This low doping level is explained by the ABA-stacking mode of I-COF which compromises the loading capacity since each I-COF monolayer is covered up by the neighboring upper and lower I-COF layers, as aforementioned.The elemental analysis data of Tb-COF are measured as C, 64.61; H, 3.90; and N, 8.55. Upon a 100% ionic exchange, the molecular composition should be Cvia their thermogravimetric analysis (TGA) curves shown in 2O, and the following three are assigned as thermal degradation of the I-COF framework. As for Tb-COF, there is no obvious weight loss of adsorbed H2O. This is because the ionic exchange procedure removes most adsorbed H2O molecules. There are three endothermic peaks at 240°C, 440°C, and 511°C, respectively, corresponding to the thermal degradation in the Tb-COF framework. At 600°C, Tb-COF weight residual is measured as 3.7% which is assigned as the metal residual . The incorporation of [Tb(PDA)3]3- component tends to move these endothermic peaks to lower temperature. Nevertheless, these endothermic peaks higher than 200°C indicate that Tb-COF is thermally stable enough for later application. Even being soaked in HCl solution (3 M), this hexagonal structure is still well-preserved, as shown in The thermal stability of I-COF and Tb-COF is investigated 33- is partially compromised since the DPA-excited state tends to transfer its energy to a surrounding lower energy level of the COF π–π* excited state, leading to the strong π–π* red emission and weak Tb(III) emission as aforementioned  upon the PA concentration of 10 μM, which shall be attributed to the strong electron-withdrawing effect of PA. The presence of PA favors Tb(III) emission and compromises π–π* red emission, showing ratiometric signals and favoring sensing application.It has been previously confirmed that Tb-COF is stable enough in acidic medium and thus can be developed for PA optical sensing. To assess the sensing performance of Tb-COF, its emission spectra upon increasing PA concentrations are considered. As shown in entioned . Given Plim is the critical value of 100% complexation, and X0 is the initial value 3]3- and PA, it is assumed that each hexagonal cage of I-COF fits only one PA molecule as well, which is a stoichiometry ratio of 1:1 as well. In this case, the emission intensity at 543 nm is analyzed via Stern–Volmer equation, described by 0 is the emission intensity without any quencher (PA), and Ks is the complexation constantAn ideal curve of 0 = 1.223 + 6.709 × 105 M−1(PA), R2 = 0.996, within (PA) the region of 0–9 μM. The limit of detection  value is calculated as 0.8 μM (15–17). Upon a higher [PA] value, a down-bending tendency is observed, indicating a 100% complexation between Tb-COF and PA.The fitting equation is obtained as I/I0 means the initial Tb-COF emission intensity at 635 nm without PA, F is the emission intensity at 635 nm, and Ks′ is the complexation constant.On the other hand, the emission intensity at 635 nm follows a non-linear decreasing curve. The residual emission intensity against quencher (PA) concentration shall be explored by another form of Stern–Volmer equation described as 0/F = 0.917 + 4.637×105 M−1(PA), R2 = 0.998, within the (PA) region of 0–8 μM. Upon a higher (PA) value, a down-bending tendency is observed, indicating a 100% complexation between Tb-COF and PA.The fitting equation is obtained as F543/I635 shows a down-bending curve with increasing (PA), instead of linear ones reported by literatures (15–17). This result indicates that the sensing mechanism of Tb-COF toward is not the energy competing between Tb-COF emissive center (635 nm) and Tb(III) ion (543 nm), although such energy competing mechanism has been reported and confirmed.The ratiometric value I0 of Tb(III) emission follows linear increasing correlation with (PA) and 2) F0/F of Tb-COF π–π* emission follows linear increasing correlation with (PA), it is assumed that the amount of Tb(III) emitter is linearly increased by (PA), while Tb-COF π–π* emission is just quenched by PA. A possible sensing mechanism is thus proposed and shown in 3]3- component trapped in Tb-COF, releasing free luminescent [Tb(PDA)3]3-. After incorporating PA in the hexagonal l cage, the π–π* emission is quenched, leading to decreased emission intensity at 635 nm. The emission decay dynamics of Tb-COF at 543 and 635 nm were recorded. Single-exponential decay patterns were observed as shown in The aforementioned analysis on working curves of Tb-COF has denied the sensing mechanism of energy competing between Tb-COF emissive center (635 nm) and Tb(III) ion (543 nm). Based on the below two facts, 1) I/I3]3- component has been released upon the presence of PA. In this case, the sensing mechanism is confirmed.For a tentative evaluation on this sensing mechanism, I-COF emission intensity against increasing (PA) is shown in +, Ca2+, Mg2+, Fe2+, Hg2+, CO32-, ACO−, Cl−, SO4−, NO3−, p-nitrobenzene, 2,4-dinitrotoluene, and 2,4,6-trinitrotoluene. It is observed from 3]3- component, so that no [Tb(PDA)3]3- emitter is released, showing stable Tb(III) emission (543 nm). Although some cationic ions such as Fe2+ and Hg2+ can quench π–π* emission (635 nm), the quenching effect is still minor. As for anionic ions, they may partially release the [Tb(PDA)3]3- emitter, leading to improved Tb(III) emission (543 nm), but π–π* emission (635 nm) is not effectively quenched. In addition, owing to the small size of these interfering anionic ions, the [Tb(PDA)3]3- emitter shall not be fully released and may co-exist in the COF hexagonal cage. As for the nitrobenzene compounds, they are neutral ones and impossible to be exchanged with [Tb(PDA)3]3-, even though they are also electron-deficient ones and quench π–π* emission (635 nm). Since no [Tb(PDA)3]3- emitter is released, no Tb(III) emission is increased. The good selectivity of Tb-COF toward PA over these interfering ions and compounds is clearer when comparing their ratiometric values (I543/I635), as shown in 3]3- emitter and quenching π–π* emission (635 nm).The selectivity performance of Tb-COF toward PA is explored by comparing its emission spectrum with those under interfering ions and compounds, including KAfter checking the sensing mechanism of I-COF toward PA, it is found that PA molecules are captured and immobilized in the COF hexagonal cage, which endows I-COF with adsorption and removal feature for PA as well. Aiming at an evaluation on the adsorption performance of I-COF, 100 mg of I-COF wad dispersed in ethanol (5 ml), then PA was added for adsorption. Later, I-COF was centrifuged, solution was extracted, and PA residual was determined by chromatography. The retrieved I-COF was sent to repeat aforementioned procedure. The total adsorbed PA amounts are listed in 33- was loaded onto the I-COF hexagonal cage via ionic exchange procedure. The resulting composite material (Tb-COF) was fully characterized by geometric analysis, IR, XRD, porosity analysis, SEM/TEM, and elemental analysis. It was found that Tb(DPA)33- was loaded into the hexagonal cage in the I-COF host with ionic exchange ratio of 41%. Tb-COF was stable enough for PA sensing as suggested by TGA and the acidic soaking experiment. The as-synthesized Tb-COF showed weak Tb(III) emission and strong red COF emission, after adding PA, Tb(III) emission was increased whereas COF emission weakened greatly, showing sensing behavior. Linear working curves were observed with good selectivity. PA molecules replaced the [Tb(PDA)3]3- component trapped in Tb-COF, releasing free luminescent [Tb(PDA)3]3-. After incorporating PA in the hexagonal cage, the COF emission was quenched. This sensing mechanism ensured a good selectivity over competing species, including cations, anions, and nitrocompounds. The adsorption and removal performance of I-COF for PA was investigated as well. For the latter experiment, the dopant adsorption capacity in COF should be improved.In this work, an ionic COF (I-COF) was synthesized, then the Tb(III) emitter Tb(DPA)


In acutely isolated rat cardiomyocytes, Nar was further explored for effects on L-type Ca2+ currents, cell contractility and Ca2+ transients by using patch-clamp technique and Ion Optix system. Network pharmacology analysis indicated that Nar improved apoptosis, mitochondrial energy metabolism, inflammation and oxidative stress. Experimental validation demonstrated that Nar decreased ROS and MDA levels and increased antioxidant activity , mitochondrial membrane potential, ATP and Ca2+-ATPase contents. Nar also markedly reduced inflammatory factor levels, apoptosis, and intracellular Ca2+ concentrations in H9c2 cells. Based on the experimental results, it is speculated that Ca2+ signals play an essential role in the process of Nar against MI. Thus, we further confirmed that Nar significantly inhibited the L-type Ca2+ currents, contractility and Ca2+ transients in acutely isolated cardiomyocytes. The inhibition of Ca2+ overload by Nar may be a novel cardioprotective mechanism. The present study may serve as a basis for future clinical research, and Nar as a Ca2+ channel inhibitor may provide new perspectives for the treatment of myocardial ischemic diseases.Naringenin (Nar) is a natural flavonoid extracted from citrus fruits with abundant pharmacological properties against cardiac diseases, but existing studies are unsystematic and scattered. The present research systematically investigates the mechanism of action of Nar in the treatment of myocardial ischemia (MI). Network pharmacology was used to analyze the relevant targets of Nar against MI as well as the biological mechanisms. The protective effect of Nar was initially assessed in H9c2 cells induced by CoCl The Traditional Chinese Medicine Systems Pharmacology Database , Comparative Toxicogenomic Database , PharmMapper (http://lilab-ecust.cn/pharmmapper/) and Swiss TargetPrediction (http://swisstargetprediction.ch/) were used to search for the target corresponding to naringenin. The standard gene names of target proteins were gained from UniProtKB (https://www.uniprot.org/). Here, search for disease-related targets in three databases using the keyword "myocardial ischemia”, including the GeneCards database (https://www.genecards.org/), Database of Gene-Disease Associations  and Online Mendelian Inheritance in Man .The 2D Structure and SMILES information of Nar were collected from PubChem (https://bioinfogp.cnb.csic.es/tools/venny/) to create Venn diagrams and obtain drug-disease intersection targets. The protein-protein interaction (PPI) network of the intersection targets was then constructed in the string database (https://string-db.org/). The “string.tsv” file was downloaded, and Cytoscape 3.6.1 was used to create a network of MI-Nar intersection targets. Finally, the Database for Annotation, Visualization and Integrated Discovery (DAVID) database (https://david.ncifcrf.gov/) was used for GO and KEGG analysis to visualize and analyze the obtained data. In addition, p values <0.01 were set to better predict and validate biological processes and mechanisms.The targets associated with MI and Nar were entered into an online Venn analysis tool (Nar (98% purity) was provided by Chengdu Alfa Biotechnology Co., Ltd. . Nar was dissolved in dimethyl sulfoxide (DMSO) to prepare the concentrations required for subsequent experiments. Penicillin/streptomycin and trypsin were purchased from Leagene Biotechnology . Phosphate-buffered saline (PBS) was provided by Solarbio Life Sciences . High-glucose Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were supplied by Gibco . Cell Counting Kit-8 (CCK-8) was acquired from Beijing Zoman Bio-technology Co., Ltd. . Collagenase type II was obtained from Worthington Biochemical . Isoproterenol (ISO) and verapamil (Ver) were provided by Hefeng Pharmaceutical Co., Ltd. . All other reagents are of analytical quality and were obtained from Sigma Chemical Company .X , ATP , Ca2+-ATPase , TNF-α , and IL-6 .All kits used here were obtained from Jiancheng Bioengineering Institute : CK , SOD , LDH , BCA , CAT , MDA , GSH-P2 incubator at 37°C. Cells were then subcultured or inoculated into 96-well plates according to experimental design.H9c2 cardiomyocytes, a subclonal cell line derived from embryonic rat myocardium, were provided by the Bluefbio . H9c2 cells were cultured in DMEM containing 100 U/ml penicillin/streptomycin and 10% FBS and maintained in a 5% CO2) is used as a common chemical hypoxic agent to simulate hypoxic/ischemic conditions in a variety of cells (2 concentrations were determined. Cells inoculated in 96-well plates were randomly divided into four groups: 1) control group (Con); 2) CoCl2; 3) L-Nar + CoCl2; and 4) H-Nar + CoCl2. Incubation with CoCl2 (600 μM) for 24 h was used to simulate an ischemic environment. H9c2 cells were pretreated with Nar (10 μM or 30 μM) for 4 h before CoCl2 treatment. Using the CCK-8 kit, the viability of the cells was determined after the experiment.Cobalt chloride  was added to the cell precipitate for suspension, and the cells were centrifuged again in similar conditions. Next, the supernatant was removed, and the cells were collected for ultrasonic disruption. BCA is used to determine the protein concentration in each. The SOD, CAT, GSH-P2 as described previously; each well was then rinsed twice with PBS. Next, using 2,7-dichlorodihydrofluorescein diacetate , we assessed the intracellular ROS levels. The green fluorescence intensity was proportional to the level of ROS. The wells of each group were added with 500 μl of DCFH staining solution, which was kept at 37°C away from light for 15 min. Afterward, the dye was discarded, and the cells were washed twice with PBS. Finally, fluorescence images were acquired by fluorescence microscopy, and the degree of fluorescence was analyzed by ImageJ software.H9c2 cells were plated in 24-well plates. The cell supernatant was discarded after treating the cells with Nar and CoCl2, H9c2 cells were washed twice with PBS and incubated with rhodamine-123  at 37°C away from light for 15 min. All experimental steps were completed according to the manufacturer’s requirements. Finally, fluorescence microscopy was used for image acquisition.As a result of treatment with Nar and CoClApoptosis levels of H9c2 cells were detected using Hoechst-33258 staining . Briefly, after drug treatment, cells were incubated with Hoechst-33258 at 37°C for 20 min and followed by two washes with PBS to remove free dye. Afterward, images were collected by using a fluorescence microscope.Briefly, cells were washed twice with PBS and stained with fluo-3, AM  for 15 min at room temperature in the dark environment, followed by two washes with PBS. After that, fluorescence microscopy was used to acquire images.ad libitum. After one week of adaptive feeding, one rat per day was used to acutely isolate cardiomyocytes, and the remaining rats were fed normally. All experimental procedures regarding animals were complied with the Guidelines of Animal Experiments from the Ethics Committee of Hebei University of Chinese Medicine.Forty adult male Sprague-Dawley rats weighing 220–250 g, 6–8 eeks, were bought from the Experimental Animal Center of Hebei Medical University . Animals are housed under standard laboratory conditions . All rats were fed animal maintenance chow  and provided with purified water available 2+-free Tyrode’s solution containing (in mM): 0.33 NaH2PO4, 1 MgCl2, 5.4 KCl, 10 Glucose, 10 HEPES, and 135 NaCl with a pH of 7.4 adjusted with NaOH. The aorta of the heart is then inserted into the Langendorff device, and the heart was perfused with Ca2+-free Tyrode’s solution for 5 min at a 37°C constant temperature device. After that, the heart was perfused with Ca2+-free Tyrode’s solution with the addition of type II collagenase (0.6 mg/ml), CaCl2 (0.03 mM), Taurine (4.4 mM) and bovine serum albumin (0.5 mg/ml) for 20–25 min. After the heart became soft, the enzymatic fluid in the heart was washed away by Tyrode’s solution without Ca2+. The heart was then placed in Krebs buffer (KB) containing (in mM): 25 KH2PO4, 40 KCl, 80 KOH, 50 Glutamic acid, 10 Glucose, 10 HEPES, 20 Taurine, 1 EGTA, 3 MgSO4, and dissected into small pieces. Finally, ventricular myocytes were suspended in KB fluid.Single ventricular myocytes were isolated from 6-8-week-old rats as previously described . After aRats were subcutaneously injected with ISO (85 mg/kg) for two consecutive days to establish a myocardial ischemia model according to previous studies . After t2+ currents. Simply, the pipette puller  was used to pull a glass pipette with a resistance of 3–5 MΩ filled with (in mM) 20 TEACL, 10 HEPES, 10 EGTA, 5 Mg-ATP, and 120 CsCl with a pH of 7.2 adjusted with CsOH. Myocardial cells were bathed in extracellular solution containing the following compounds (mM): 10 Glucose, 10 HEPES, 1.8 CaCl2, 2 MgCl2 and 140 TEACL with a pH of 7.4 adjusted with CsOH. This solution allows to reduce the interference from other currents. The electrodes were gently pressed onto the cell surface. The cell was aspirated and formed a high resistance seal at a zero potential difference between the inside and outside of the electrode. Axon patch 200B Amplifier  was filtered at 2 kHz and recorded the Ca2+ currents. Analyses of the data were carried out using the pCLAMP 10.7 software .Isolated cardiomyocytes were investigated under a whole-cell patch clamp to record Ca2+ transients were recorded as described  and field stimulation was given at 0.5 Hz (duration 2-ms) using a cell stimulator .Cardiomyocyte contractility and Caescribed . Briefly2+ transients were detected simultaneously with contraction of the cardiomyocytes. Experimental steps were performed in the dark due to the light sensitivity of the fluorescent Ca2+ indicator. Cardiomyocytes were loaded with fluorescent Ca2+ indicator fluo2-AM (2 mM) for 10 min. Cells were stimulated with a field intensity of 0.5 Hz and passed alternately through 340 and 380 nm filters (bandwidth of ±15 nm). Finally, the emitted fluorescence was observed at 510 nm.Cap < 0.05 was considered statistically significant. Clampfit 10.7 (Molecular Devices) was used to analyze the ICa-L data, and Boltzmann functions were used to fit the steady-state activation and inactivation of ICa-L.Data were processed and analyzed using Origin 7.5 software (OriginLab Corp.). Statistical analysis of the data was conducted using one-way analysis of variance (ANOVA) followed by Bonferroni correction for multiple comparisons. Data were expressed as the means ± SEM. ADME-related information of Nar was searched by TCMSP. OB is the oral bioavailability of the drug and DL is the similarity of the drug, which are important for the screening and evaluation of the drug. The OB and DL of Nar were 59.29% and 0.21, respectively, which met the lower limit.15H12O5, MW:272.27). A total of 235 targets of Nar were obtained from TCMSP, CTD, PharmMapper and Swiss TargetPrediction databases. Meanwhile, a total of 1,587 targets of MI were obtained from GeneCards, DisGeNET and OMIM databases. The Venn diagram showed that 130 potential targets had relationships with Nar and MI , cellular component (CC), and molecular function (MF) consisted of 241, 17, and 31 items, respectively. BP analysis showed that most of these targets are closely related to the regulation of apoptotic processes, redox, response to hypoxia, inflammatory response, regulation of cell proliferation, and oxidative stress response. CC analysis indicated that these targets were distributed in the cytosol, mitochondria, and endoplasmic reticulum. MF analysis revealed that these targets were associated with protein binding, ATP binding and enzyme binding. KEGG enrichment analysis suggests that the majority of pathways mentioned here are closely related to apoptosis and inflammation . The res2. Nar itself had almost no cytotoxic effect on H9c2 cells in the concentration range of 1–100 μM; the strongest protective effect on cells was at 30 μM of Nar (2 group versus the Con group (p < 0.01). Treatment of H9c2 cells with 10 or 30 μM Nar attenuated 600 μM CoCl2-induced cell damage (p < 0.05 or 0.01).The protective effect of Nar was initially assessed based on the survival of H9c2 cells induced by CoClM of Nar . Therefo2 treatment (p < 0.01), whereas Nar treatment reduced the release of CK and LDH versus the CoCl2 group (p < 0.01 or 0.05).We assayed the release of CK and LDH in cells  to furth2 treatment (p < 0.01), but the fluorescence level was significantly reduced after pretreatment with different doses of Nar (p < 0.01)  .X, SOD, CAT, and MDA. The concentrations of GSH-PX, SOD, and CAT were markedly reduced after exposure to CoCl2 (p < 0.01), but these parameters were reversed after pretreatment with Nar (p < 0.01 or 0.05). Meanwhile, the content of MDA was increased after exposure with CoCl2 (p < 0.01). The MDA level in H9c2 was reduced after pretreatment with Nar (p < 0.01 or 0.05) (We next measured the intracellular levels of GSH-Por 0.05) . These r2 treatment, lower levels of green fluorescence were observed in H9c2 cells. (p < 0.01), however, the fluorescence intensity was significantly enhanced after pretreatment with Nar. The results indicated that Nar can maintain the integrity of mitochondria.Mitochondrial dysfunction is closely associated with myocardial ischemia . We eval2+-ATPase activity is an indirect reflection of the change in ATP content (2+-ATPase were increased to different extents in H9c2 cells (p < 0.1 or 0.5). However, the levels of ATP and Ca2+-ATP in the cells were significantly decreased after CoCl2 treatment (p < 0.1)  .2-treated H9c2 cells (p < 0.01). However, IL-6 and TNF-α levels were significantly decreased after pretreatment with Nar (p < 0.01 or 0.05).To identity the anti-inflammatory effects of Nar in H9c2, we next evaluated the levels of IL-6 and TNF-α . IL-6 an2 (p < 0.01). But the fluorescence intensity was notably diminished after pretreatment with Nar (p < 0.01 or 0.05)  .2+ concentration in H9c2 cells. The fluorescence intensity of Ca2+ was significantly enhanced after CoCl2 treatment (p < 0.01), however, the levels of fluorescence were markedly reduced after pretreatment with different doses of Nar (p < 0.01) (2+ concentrations.We confirmed that Nar reduces the increase of Ca < 0.01) . It was Ca-L.Two types of voltage-dependent calcium channels (L-type and T-type) are present in cardiac myocytes. T-type calcium currents are negligible in most ventricular myocytes, so calcium currents are usually referred to as L-type here . The L-tCa-L was measured under control conditions, followed by exposure to 100 μM Nar and washout . ICa-L was restored after washing off Nar with external solutions, thus suggesting that the effect of Nar on ICa-L was not caused by running down. The present results indicate that the effect of Nar on Ca2+ currents is largely reversible.I washout . Nar (10Ca-L in a concentration-dependent manner (y = A2 + (A1—A2)/[1 + (x/x0)p]. Here, y is the response, A1 and A2 are the maximum and minimum response, respectively, x is the drug concentration, and p is the Hill coefficient. The inhibition of ICa-L in myocardial cells (n = 8-10) treated with 1, 3, 10, 30, and 100 μM Nar were 2.50 ± 0.31%, 13.14 ± 2.18%, 23.22 ± 2.67%, 34.55 ± 2.59%, and 75.10 ± 3.07%, respectively. The half-maximal inhibition (IC50) was 41.493 μM.It was been found that Nar inhibited It manner . LogistiCa-L was recorded with a steady-state activation protocol along with control, Nar  and Ver conditions   of 7.69 ± 0.37 mV for control, -13.72 ± 0.34 mV with a k value of 7.49 ± 0.32 mV for Nar of 3 μM, and -15.11 ± 0.41 mV with a k value of 7.34 ± 0.37 mV for Nar of 30 µM. Values of V1/2 for the steady-state inactivation were -26.17 ± 0.79 mV with a k value of 5.99 ± 0.72 mV for control, -27.64 ± 0.73 mV with a k value of 6.03 ± 0.68 mV for Nar of 3 μM, and -29.00 ± 0.34 mV with a k value of 6.11 ± 0.32 mV for Nar of 30 µM. The results showed that Nar had no significant effect on steady-state activation and inactivation of ICa-L.We further documented the activation and inactivation gating properties of Cad 30 μM) . Values p < 0.01)  represents cellular relaxation or Ca2+ reuptake. Compared to the Basal, 100 µM of Nar enhanced the TP by 21.64 ± 8.83%, and decreased the TR by 21.43 ± 8.43% (p < 0.05) (We next determined the effects of 100 μM Nar on the contractility of cardiomyocytes. The representative track of cell shortening before and after administration of Nar (100 µM) was presented in  < 0.01) . The tim < 0.05) .2+ transients before and after the administration of Nar are presented in 2+ transients by Nar of 100 μM was 61.00 ± 2.91% (p < 0.01). The results showed that Nar partially and reversibly reduced the transient of Ca2+ currents in cardiomyocytes.Changes in cellular CaCurrently, ischemic heart disease has become a huge threat to human health and the global economy H. . Naringevia the Venn analysis tool to perform enrichment analysis. The top 10 core targets for Nar treatment of MI are MAPK3, TP53, SRC, AKT1, STAT3, HSP90AA1, RELA, ESR1and MAPK8 (degree >17). Of these, HSP90AA1 can regulate mitochondrial function to protect the heart  preventing hemoglobin from binding to oxygen, thus simulating a hypoxic environment and producing a series of reactions similar to those under hypoxic conditions. Compared to low oxygen-induced hypoxia and the use of other hypoxia mimics, CoCl2 can strongly stabilize hypoxia-inducible factor (HIF)-1α and HIF-2α under normoxic conditions, which can last for several hours  , which allows Cam the SR . We foune of Nar . Howeverroponin) . Future roponin) . The cur2+ currents, cell contractility, and Ca2+ transients. These inhibitory effects are essential mechanisms for Nar protection against myocardial ischemic damage.Calcium overload is thought to be a predisposing cause of myocardial ischemia . MyocardXenopus oocytes (The current study was devoted to exploring the protective effects and mechanisms of Nar against MI, mainly focusing on calcium channels. However, it cannot be excluded that Nar affects other ion channels in the long term. Previous studies have shown that Nar activates mitochondrial potassium channels in dermal fibroblasts and blocks HERG potassium channels in  oocytes . In addi oocytes . Therefo2+ overload in isolated cardiomyocytes. The present research provides comprehensive experimental evidence for the pharmacological effects of Nar, which offers a new perspective on Nar in the treatment of ischemic heart disease.This work is to systematically explore the potential targets of Nar for the treatment of MI using network pharmacology. It was demonstrated that Nar attenuated MI-induced oxidative stress, mitochondrial dysfunction, inflammation and apoptosis. Most importantly, the current study indicated that Nar exhibited cardioprotective properties by inhibiting LTCCs thereby reducing Ca