Salt sensitivity of blood pressure (BP) refers to an increase in BP following an increase in dietary salt, which is associated with increased incidence of cardiovascular disease and early death. However, decreased sodium intake also increases mortality and morbidity. Inverse salt sensitivity (ISS), defined as a paradoxical increase in BP on a low-salt diet, about 11% of the population, may be the cause of this phenomenon. The epithelial sodium channel (ENaC) is a major regulator of sodium reabsorption in the kidney. In this study, human renal tubular epithelial cells (hRTC) were cultured from the urine of phenotyped salt study participants. αENaC expression was significantly lower in ISS than salt resistant (SR) hRTC, while ENaC-like channel activity was dramatically increased by trypsin treatment in ISS cells analyzed by patch clamp. αENaC expression was also decreased under high-salt treatment and increased by aldosterone treatment in ISS cells. Moreover, the αENaC variant, rs4764586, was more prevalent in ISS. In summary, αENaC may be associated with ISS hypertension on low salt. These findings may contribute to understanding the mechanisms of ISS and low salt effect on morbidity and mortality. It acts as a key regulator of sodium reabsorption in distal convoluted tubule (DCT), connecting tubule (CNT), and principal collecting duct (CD) [The amiloride-sensitive epithelial sodium channel (ENaC) is located mostly in the apical membrane of epithelial cells of the lung, distal nephron, distal colon, and ducts of exocrine glands . As a noia cells ,3,4. ENaia cells ,5. Each ia cells ,7. Renaluct (CD) ,9. In thuct (CD) ,11.+ wasting [+ or high K+ feeding [+ restriction [+ load [+ balance, we hypothesized that ENaC may differentially regulate Na+ transport in salt resistant (SR) and ISS individuals. In order to test this hypothesis, we measured the presence of ENaC in CD13-positive renal cells collected from the urine of our dietary salt study participants who were ultimately phenotyped for their BP response to randomized transitions between week-long high (300 mEq) and low (10 mEq) Na+ diets [Abnormalities in ENaC expression or function are associated with human pathology. Genetic deletion or loss-of-function mutations in any ENaC subunit cause pseudohypoaldosteronism type 1 (PHA-1) which is characterized by Na wasting ,13,14,15 wasting ,17,18. C feeding . Howevertriction . The Gentriction . Salt se [+ load ,23,24,25 [+ load . As ENaCn = 280) were grouped into three classes, depending on the BPs obtained after 14 days of diets prepared by a dietician in the University of Virginia Medical Center containing 10 mEq Na+ (as NaCl) and 300 mEq Na+ (+ diet minus the MAP on the last day of the low Na+ diet. The subjects were classified into SR (64% of participants) if they had an increase in MAP of <7 mmHg on 300 mEq Na+ intake and a decrease in MAP of <7 mmHg on 10 mEq Na+ intake [+ intake and a decrease in MAP ≥ 7 mmHg on 10 mEq Na+ intake [+ intake and an increase in MAP ≥ 7 mmHg on a 10 mEq Na+ intake [Specimens were collected from volunteer participants enrolled in an Institutional Review Board-approved dietary salt study . The par mEq Na+ . The BP + intake ; SS  at 37 °C in 5% CO2–95% air atmosphere [Human renal tubule cells (hRTCs) were collected from urine freshly voided by participants on regular diet in our clinical study as described in our previous study . The celmosphere ,28. In amosphere . We havemosphere .7) and then trypsinized, filtered using a 30-µm filter, and centrifuged at 1000× g for 3 min. The pelleted cells were rinsed with cold MACS buffer , and re-centrifuged at 1000× g for 3 min. The supernatant was discarded, 100 µL of MACS Buffer  was added, and the suspension was transferred to a 1.5-mL tube with 5 µL of CD13-PE . After mixing, the cells, protected from light, were incubated at 4 °C for 20 min, and then 1 mL of MACS Buffer was added, mixed well, and centrifuged at 1000× g for 3 min. The pelleted cells were then resuspended in 100 µL MACS Buffer mixed with 10 µL anti-PE magnetic beads (Miltenyi Biotec) for 20 min at 4 °C. The cells were rinsed, collected and finally resuspended in 500 µL MACS Buffer and transferred to a 5 mL tube that was kept on ice and protected from light. The cells were purified to near homogeneity using an AutoMACS (Miltenyi Biotec) for selection, using a single column. Positive and negative fractions were measured by flow cytometry .Cells were grown in 15-cm dishes to 85% confluence . Images were acquired using a 20× and a 60× UPlanSApo water immersion objectives for tissue section and cell imaging. The microscope was controlled by Metamorph software , which can stich single images together to obtain a view of a larger number of cells.2 incubator for 24 h. Then, the medium was changed and the cells incubated for an additional 24 h. Non-targeting siRNA  was used as control. At the end of the additional 24 h of incubation, the cells were harvested and prepared for Western blotting.The hRTCs were plated in 6-well plates and grown to 50% confluent. Human αENAC siRNA  was mixed with Lipofectamine RNAiMAX  for 20 min at room temperature. The mixture was then added to the cells at a final siRNA concentration of 100 nM. The cells were incubated at 37 °C in a 5% COThe hRTCs  were lysed in M-PER mammalian protein extraction buffer  and sonicated. The total protein in supernatant was measured by Bradford assay , and the proteins in the samples were separated by SDS-PAGE (4–20% gradient gel). Western blotting was performed, as previously described . Rabbit Three αENaC SNPs, rs11614164, rs3741914, and rs4764586, which are associated with salt-sensitive BP, were chosen for genotyping  the huma2, 10 HEPES/Tris, pH 7.3; trypsin (5 μg/mL) was added in order to increase ENaC activity. In one set of experiments Li+ was replaced by Na+ in the pipette solution. Extracellular bath solution (in the chamber) contained (mM) 150 NaCl, 5 KCl, 5 glucose, 1 CaCl2, 2 MgCl2, 10 mM HEPES, pH 7.3. To quantify the level of channel activity, we used ClampFit software processing, and the following equation was used to define Po: Po = I/Ni, where Po—channel open probability, N—number of working channels in the patch, I—the mean current determined from the amplitude histograms, i—unitary current amplitude.Ion currents through single channels were recorded using cell-attached configuration of patch-clamp technique, based on Axopatch 200B amplifier, Analog-Digital Interface Digidata 1550B  and controlled by pClamp 10.7 software . A BessehRTCs from SR and ISS subjects were plated in 96-well glass bottom plates, and then treated overnight with 1 μM aldosterone at 37 °C . The hRTCs were fixed and stained with mouse anti-mineralocorticoid receptor antibody  and αENAC . Montage images of each well were obtained under 20× using a Cytation 5 image reader automatically and analyzed by Gen5 software  using MR (green) as the primary mask and αENAC (red) as the secondary mask. Cell number was counted and mean value of the fluorescence density (relative fluorescence unit RFU) was used to conduct analysis. Plasma aldosterone and plasma renin activity (PRA) were measured using clinically approved diagnostic tests available through the UVA clinical laboratory.t-test was used for two-group comparisons. Chi-square test was used for SNP analysis. p < 0.05 was considered as significant for all analyses.Data were expressed as means ± SE. Comparisons within and among groups (>2) were analyzed by repeated-measures or factorial ANOVA, respectively, followed by Tukey’s (comparing every mean with every other mean), Dunnet’s (comparing each mean with the other mean in that row), or Sidak’s post hoc test (comparing each mean with the control mean in that column) using GraphPad Prism 7.0 software . Student’s +-Cl− co-transporter (NCC)-positive cells [Many types of cells along the nephron were exfoliated into the urine. Two distinct types of cell colonies were observed. Type I cell colonies were round and small, with a fast growth rate and have clearly defined edges. The cells in the Type I colonies exhibited a dense growth pattern i, ii of A. The ceve cells  . To confirm the expression of αENaC in urine-derived hRTCs, αENaC expression was knocked down by siRNA in 3 SR and 3 ISS cell lines. Western blot analysis confirmed that a single band of human αENaC with apparent molecular size between 75 and 100 kDa was present in all cell lines and reduced in the knockdown cells . βENaC appeared as a band with expected molecular weight between 75 and100 kDa, while γENaC appeared as two bands between 75 and 100 kDa , but decreased it in ISS cells   D,E, indirev = 34.1 ± 1.5 mV in SR cell and 10.3 ± 0.8 pS and Erev = 33.6 ± 5.5 mV in ISS cells, + was the main cation in the pipette solution vs. Li+. The current-voltage dependency (IV) showed lower single channel conductance of ENaC-like channels in SR cells with Na+ in the pipette solution compared to Li+ .The activity of single ENaC channel in the plasma membrane of urine-derived hRTCs was measured with the cell-attached configuration of the patch-clamp technique. The ENaC-like channels had a similar conductance in SR and ISS cells   (+) [n = 9/group, p < 0.05; αENaC: ISS VEH 4561 ± 68 vs. ISS Aldosterone 4834 ± 52, n = 9/group, p < 0.01, two-way ANOVA, Sidak’s post hoc test)  B, indicaoc test) B. Moreovoc test) . We thenest) (+) . After aoc test) .2 = 9.73, p < 0.05)  . However < 0.05) . Urine-d+ homeostasis. The ENaC mediated Na+ reabsorption occurs predominately in collecting duct because of its abundant expression in that nephron segment. Using urine-derived renal tubule cells (RTC) and human kidney tissue sections, our current studies demonstrate that α, β, and γENaC are present in urine-derived RTC, including PTC. The immunostaining was performed by two sets of antibodies, especially the StressMarq antibodies developed by Dr. Mark Knepper [Renal ENaC plays an important role in the regulation of whole-body Na Knepper . Both α + current [Xenopus, α, β, and δ ENaC can form homomeric functional channels that show amiloride sensitive currents, and channels containing two subunits also respond to shear force [RNA-seq data in mouse and rat demonstrate that αENaC is more ubiquitous and plentiful than the other two subunits ,40. Even current . Some va current . Moreove current ,46. In Xar force . Therefo+-replete diet compared with the rats on low-Na+ diet [Differential regulation of sodium balance contributes to an individual’s “personal salt index” . InverseNa+ diet . Single-Aldosterone is dramatically increased under low salt conditions . It alsoαENaC variants were reported to be associated with salt sensitivity of BP in a Chinese GenSalt study . Those wOthers have also demonstrated the utility of urine-derived cells. For example, the first successful culture of exfoliated urinary cells was from newborn infants reported by Sutherland and Bain , and sub+ homeostasis and also exhibit necrotic features. Another limitation is the sample size of urine cell lines, which is dependent on participant recruitment and urine cell culture. Furthermore, characterization of these urine-derived renal tubule cells should be performed in later passages because the cells have stem-cell characteristics and may de-differentiate after being subcultured many times [+ increases or decreases ENaC expression. Further, the differential regulation of ENaC between ISS and SR also need to be further addressed. For example, Mammalian Ste20 kinase 3 (MST3) may be involved in regulating ENaC expression in these hRTCs, which maintain BP through inhibiting ENaC expression in mice [A limitation of our studies is that we did not determine the effect of high and low-salt diets on ENaC expression and activity in renal tissue. Renal tissue cannot be obtained from normal individuals as IRB approval cannot be obtained for experimental renal biopsies. Discard kidneys from transplant services have proved not to exhibit normal physiological responses related to Nany times . Physiolny times . Future  in mice ,67.


Nowadays, IVD acid environment variation which accelerates the pathological processes mentioned above arouses researchers' attention. KAN0438757 (KAN) is an effective inhibitor of selective metabolic kinase phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) that has both energy metabolism reprogramming and anti-inflammatory effects. Therefore, a potential therapeutic benefit of KAN lies in its ability to inhibit the development of IVDD. This study examined in vitro KAN toxicity in NP primary cells (NPPs). Moreover, KAN influenced tumor necrosis factor-α (TNF-α) induced ECM anabolism and catabolism; the inflammatory signaling pathway activation and the energy metabolism phenotype were also examined in NPPs. Furthermore, KAN's therapeutic effect was investigated in vivo using the rat tail disc puncture model. Phenotypically speaking, the KAN treatment partially rescued the ECM degradation and glycolysis energy metabolism phenotypes of NPPs induced by TNF-α. In terms of mechanism, KAN inhibited the activation of MAPK and NF-κB inflammatory signaling pathways induced by TNF-α and reprogramed the energy metabolism. For the therapeutic aspect, the rat tail disc puncture model demonstrated that KAN has a significant ameliorated effect on the progression of IVDD. To sum up, our research successfully authenticated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect.Intervertebral disc (IVD) degeneration (IVDD) is a characteristic of the dominating pathological processes of nucleus pulposus (NP) cell senescence, abnormal synthesis and irregular distribution of extracellular matrix (ECM), and tumor necrosis factor- As a result of IVDD, low back pain and severe spinal disorders, including lumbar disc herniation and spinal stenosis, are common . Due to ammation  and the ammation –8, no maammation –11, haveα production and expression of other inflammatory mediators [However, some emerging but interesting perspectives have aroused researchers' attention in the past few years. One of these hot issues, acid environment variation of degenerated IVD, similar to hypoxia environment change, has been demonstrated to influence some processes of IVDD , 13. Speediators . Thus, tκB and MAPK pathways) and blocking glycolysis of osteoclasts at the same time [PFKFB3 is a key glycolysis rate-limiting enzyme. Due to its strong kinase activity, stabilized expression of PFKFB3 can elevate the rate of glycolysis, thereby promoting the blood vessel sprouting, affecting tumor angiogenesis . Therefoame time .KAN is the latest inhibitor of selective metabolic kinase PFKFB3, more effective than PFK15, that has been identified as a potential cancer treatment strategy . Therefoβ, phospho-IKKα/β, IκBα, phospho-IκBα, P65, phospho-P65, and β-actin and the secondary antibody were purchased from Cell Signaling Technology . All the antibodies mentioned except β-actin are rabbit anti-mouse.The selective metabolic kinase PFKFB3 inhibitor KAN0438757 (KAN) was purchased from MCE , dissolved in dimethyl sulfoxide (DMSO) as a 5 mM stock solution, and stored at −80°C. A final concentration of DMSO less than 0.1% was used to reduce cytotoxicity. Fetal bovine serum (FBS) and penicillin/streptomycin were bought from Gibco BRL . Minimal essential medium (DMEM) was bought from Hyclone . The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Japan). The prime script RT Master Mix kit and TBGreen ® Premix Ex Taq ™ kit were both obtained from Takara Biotechnology . The XF Cell Mito Stress Test (for OCR) Kit, XF Glycolysis Stress Test (for ECAR) Kit, XFe96 FluxPak mini, XF DMEM Base Medium, XF 1.0M Glucose Solution, 100 mM XF Pyruvate Solution, and 200 mM XF Glutamine Solution were obtained from Agilent . The primary antibodies against MMP9, MMP13, and PFKFB3 were purchased from Abcam . Primary antibodies including ERK, JNK, p38, phospho-ERK (Tr202/Tyr204), phospho-JNK (Tr183/Tyr185), phospho-p38, IKK2, and 5% CO2 incubator. Then, the mixture was centrifuged, and cells were separated, resuspended, and seeded (DMEM with 10% FBS and 1% penicillin-streptomycin) in a culture plate placing on 37°C, 21% oxygen, and 5% carbon dioxide incubator.Pentobarbital sodium 50 mg/kg (body weight) was injected intraperitoneally into 4-week-old SD rats for euthanasia. After that, the NP tissues were obtained from the caudal discs (Co1–Co6) and then digested for 2 hours by 0.25% type II collagenase at 37°C, 21% O3 cells per well in presence of various concentrations of KAN (control (0), 156.25, 312.5, 625, 1250, 2500, and 5000 nM) and cultured for 24 h. As for proliferation, 96-well plates were seeded with NPPs in triplicate at 2 × 103 cells per well for 24 hours first. Then, KAN concentrations of control, 156.25, 312.5, 625, 1250, 2500, and 5000 nM were added into the wells for 24, 48, 72, and 96 hours. Cells were incubated at 37°C for 2 hours with 10 μL of the CCK-8 reagent after each experimental period. The optical density values were measured on the Infinite M200 Pro multimode microplate reader using a spectrophotometer at 450 nm .The cell proliferation and toxicity effects of KAN on NPPs were measured using the CCK-8. More specifically, to determine toxicity, the NPPs were seeded triplicately into 96-well plates at 8 × 10μl medium micromasses containing approximately 1.2 × 105 NPPs were seeded in the middle at the bottom of a 24-well plate first. Then, the plate was placed in a 37°C, 21% O2, and 5% CO2 incubator for 90 min for the attachment of NPPs to the bottom. After that, 500 μL of MEM/F12 medium (2% FBS and 10 ng/mL of Insulin Transferrin Selenium (ITS)) was added into the plate. The medium was changed every two days; These micromasses were stained with Alcian blue at the day 7.To assess the ECM secretion ability effect of KAN, 10 3/well  for the first 24 hours. Then, the cells were treated with control, TNF-α, or TNF-α plus 2.5 μM KAN for the next 24 hours. The measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were carried out according to standard protocols, and the results were detected with the Seahorse XF-96 Flux Analyzer (Seahorse Bioscience), as previously described [μM oligomycin, 2.5 μM FCCP, rotenone, and 0.5 μM antimycin A were used to stimulate cells. In the EACR assay, 10 mM glucose, 2 μM oligomycin, and 50 mM 2-DG were used to stimulate cells. Additionally, the OCR and ECAR changes were used to calculate OXPHOS characteristics as well as glycolysis parameters  fix, and 10 min 0.2% Triton X-PBS permeabilization were performed on NPPs. The phospho-p65 primary antibody  was then incubated with cells overnight at 4°C after 10% goat serum blocking. After three times washing with PBS on the next day, the cells were incubated at dark with the Alexa Fluor 594 conjugate anti-rabbit secondary antibody for 1 hour . The cell nucleus was shown by 4′,6-diamidino-2-phenylindole (DAPI) after 3 min incubation . Finally, digital fluorescence results were observed, and images were captured by the Leica fluorescence microscope .In a 6-well plate, 2 × 105 cells/well NPPs were cultured in DMEM on 6-well plates for 24 hours. The NPPs were then treated with control, TNF-α, or TNF-α plus 2.5 μM KAN for 1 day. As for the effect of KAN on the phosphorylated proteins (short-time activated), 5 × 105 cells/well NPPs were cultured in DMEM on 6-well plates for 24 hours. The treatment group cells were then pretreated with 2.5 μM KAN for 2 hours with serum-free DMEM. 2 hours later, 10 and 30 min of TNF-α (20 ng/mL) stimulation were performed on the TNF-α group and the treatment group. After that, RIPA lysis buffer plus phosphatase and protease inhibitors  were used to, respectively, extract total cellular proteins at indicated time points. According to the standard protocol of the bicinchoninic acid protein quantification kit, protein concentrations were then quantified . Standard western blot experiments were performed as previously described [β-actin, 1 : 1000; ERK, 1 : 1000; JNK, 1 : 1000; p38, 1 : 1000; IKKβ, 1 : 1000; IκBα, 1 : 1000; P65, 1 : 1000; MMP9, 1 : 1000; MMP13, 1 : 1000; PFKFB3, 1 : 1000; p-JNK, 1 : 1000; p-ERK, 1 : 1000; p-p38, 1 : 1000; p-IKKα/β, 1 : 500; p-IκBα, 1 : 1000; p-P65, 1 : 500) incubated overnight at 4°C. Membranes were extensively washed in TBST for three times on the next day. The secondary antibody ) was subsequently incubated with the membranes for 1 hour at RT in the dark. Finally, the LI-COR Odyssey fluorescence imaging system was used to detect the reactivity , and the ImageJ software was used to measure the grey values of each protein .To figure out the effect of KAN on the catabolic or anabolic proteins, 3 × 10escribed . Primary5 cells/well NPPs were cultured in DMEM on 6-well plates. 24 hours later, the NPPs were cultured with control, TNF-α, or TNF-α plus 2.5 μM KAN for 24 hours. Total RNA was extracted using the Axygen RNA Miniprep Kit  when the culture was completed. The Prime Script RT Master Mix kit was then used to reverse transcript the RNA to cDNA. Standard PCR (RT-qPCR) analyses were then performed as described previously [ΔΔCT method, relative mRNA expression levels were calculated and normalized to the β-actin expression. All primer pairs  staining and safranin O-fast green staining were then processed for histological assessment under standard laboratory protocols. All sections were captured using the high-quality microscope (Leica DM4000B). The histological score was evaluated based on the standard evaluation of 5 categories of degenerative changes [After 4% paraformaldehyde fixing for 48 hours, IVD tissue samples were decalcification in the 10% EDTA for 21 days and then embedded into paraffin blocks. After being subjected to 5  changes . For immβ and anti-collagen 2; dilution 1 : 100) , secondary antibody incubation, and nuclear staining were performed on these slides. The high-quality Leica DM4000 B epifluorescence microscope (Leica Microsystems GmbH) was used to observe and photograph the digital fluorescence images, and the ImageJ software was used to measure the IOD value.Slices were processed of graded xylene deparaffinization, graded alcohol solution hydration, antigen retrieval, permeabilization, and blocking before immunofluorescent staining. Then, primary antibody incubation (anti-interleukin- (IL-) 1Cabinet X-ray imaging and irradiation systems  were used to visualize the X-ray imaging of IVDs. Under identical acquisition parameters and imaging conditions, digital images were used to capture at 45 kVp. The disc height index of these IVDs was measured and normalized to sham group . The calμL vehicle, low-dose KAN (2.5 μM), and high-dose KAN (5 μM). After incision suture and 4 weeks of recovery, all experiment rats were sacrificed. The tails were extracted and cleaned of soft tissues and then fixed in 4% PFA.The male Sprague–Dawley rats  used in this research were carefully housed . When rats were housed to the age of eight weeks old, pentobarbital sodium (5 mg/100 g body weight) was used to anesthetize them by intraperitoneal injection before surgical procedures. The iodinated polyvinylpyrrolidone was first used for sterilization, and then, a ventral longitudinal skin incision was made over the tail. The revealed coccyx vertebrae (Co) 3–7 intervertebral discs were selected for experiments . Surgical exposure was repeated after one week recover under the sutured incision state, and Co4/5, Co5/6, and Co6/7 IVDs were separately injected with 50 All animal experiments were authorized by the Institutional Animal Care and Ethics Committee of the Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine. All animals were operated in accordance with the Guidelines for Animal Treatment of Shanghai Jiaotong University and the principles and procedures of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.t-test, or one-way ANOVA with Tukey's post hoc test. Moreover, significant differences in ordinal data between study groups were assessed by the Friedman test with Dunn's post hoc test. Statistical significance was calculated to be at no significant (ns); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. All results were calculated and analyzed by Prism 8 .All data were expressed in the form of mean ± standard deviation. The comparison between the experimental group and the control group was conducted by the 2-tailed, unpaired Student's μM) for cytotoxicity analysis, and no cytotoxicity was found in KAN under the concentration of 5 μM (μM) for 24, 48, 72, and 96 hours. The number of NPPs with any concentration of KAN has no significant variations compared with the control (0) group at any time point  group compared with the upregulated expression in the TNF-α group  and block the glycolysis of osteoclasts simultaneously [α stimulation. After that, we observed the classical NF-κB signaling pathway transduction factor change, including the inhibitor of nuclear factor kappa B kinase (IKK), an inhibitor of nuclear factor kappa B (IκB), and NF-κB (p65). As we expected, the IKK, IκB, and p65 phosphorylation were promoted with TNF-α induction while reversed after KAN treatment  induced by puncture of rat tail intervertebral disc, and Co 3-4 was used as the sham operation group. Different concentrations of KAN were separately administered one week after injury to Co 5-6 and Co 6-7, and vehicle was injected to Co 4-5. As shown by 4 weeks' X-ray detection after treatment, the osteophyte of the vehicle group was obviously observed more than other groups. Moreover, vehicle group's disc height index (DHI score) was significantly lower than the sham group, while the KAN treatment memorably reversed the IVD height in a dose-dependent manner  during metabolic bone disease osteoporosis [IVD degeneration (IVDD) is one of the most common reasons for lumbar disc herniation, spinal stenosis, and subsequent lower back pain , which hoporosis , our stuβ (IL-1β) and tumor necrosis factor-α (TNF-α), identified as a major risk factors for decreased NPP activity and extracellular matrix loss (including aggrecans) [α-induced NPP dysfunction and ECM degradation were selected to be studied in our present research. We demonstrated that nontoxic concentration KAN treatment could strongly reverse TNF-α-induced degradation of ECM. Specifically, after TNF-α induction, the upregulated MMP family  mRNA expression related to catabolism [α stimulation. In general, we demonstrated the rescue effect of KAN on ECM degradation. The underlying mechanisms were also revealed. Although a lot of signaling pathways, such as NF-κB, phosphatidylinositol-3-kinase (PI3K), MAPK, and Wnt signaling pathways, had been reported to mediate ECM metabolism or cell apoptosis after inflammation stimulation in the NPPs [κB and MAPK signaling pathways that were reported on PFK15 research. According to the report about the decisive role of TNF-α-induced NF-κB signaling pathway activation and subsequent p-p65 nuclear translocation in the onset and development of IVDD [κB signaling pathway activation and p-p65 nuclear translocation. Similarly, the repressing effect of KAN on the MAPK signaling that is important for ECM synthesis and degradation [α-induced conventional NF-κB and MAPK signaling pathways to alleviate the ECM degradation.Due to the properties of inflammatory cytokines, particularly interleukin 1grecans) , TNF-α-itabolism  and the tabolism  both partabolism  showed tthe NPPs –41, we m of IVDD , 43, we radation  was alsoα-induced NPPs. At the same time, KAN treatment partly reversed the reduction of spare respiratory capacity and maximal respiration and the increase of glycolysis rates and glycolytic capacity. All in all, our studies firstly corroborated KAN's energy metabolism reprogramming effect on TNF-α-induced glycolysis metabolic phenotype on NPPs.A more attractive role of KAN is the potential of reprogramming energy metabolism. Acid environment variation caused by NPPs aggravated glycolytic metabolism phenotype during IVDD had been demonstrated to enhance the lactate production and its efflux to ECM, which promoted the activation of NLRP3 inflammasome and the NPP extracellular matrix degeneration . The terin vivo. In our X-ray examination, the reduced DHI score of the vehicle group was memorably reversed in the high-dose KAN treatment group. Moreover, our H&E staining and safranin O-fast green staining showed more closer IVD structure of KAN treatment groups to the sham group, compared with the disordered structure of the vehicle group. Furthermore, immunofluorescence staining results manifested the inflammatory factor repression and ECM reservation effect of KAN in vivo. To sum up, these results demonstrated that KAN intervention is a promising potential therapeutic strategy for IVDD.Finally, we identified the therapeutic potential of KAN on the rat tail disc puncture-induced IVDD model κB and MAPK) remains to be revealed, and whether and how KAN influences other signaling pathways, such as PI3K, and Wnt signaling pathway is still unclear. Secondly, the ER stress [However, as the original research exploring the effect of KAN on NPPs and IVDD, we still have many limitations. Firstly, the intermediator between KAN and inflammation signaling pathways (NF-R stress , acid-seR stress , 47, andR stress , 49 are R stress . AccordiR stress , 23, theAnyway, our research successfully demonstrated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect, which provided a fresh direction for researchers focusing on the influence of the IVD environment during the development of IVDD Figures . Moreove


The encapsulates of carrot waste oil extract improved the antioxidant properties of durum wheat pasta. The aim of this research was to study the kinetics of carotenoids degradation in the freeze-dried (FDE) and spray-dried (SDE) encapsulates of carrot waste extract during storage at four different temperatures  up to 413 days by HPLC. Carotenoids levels decreased as a function of time and temperature, following zero-order kinetics. At 4 °C carotenes were stable for at least 413 days, but their half-lives decreased with increasing temperatures: 8–12 months at 21 °C; 3–4 months at 30 °C; and 1.5–2 months at 37 °C. The freeze-drying technique was more effective against carotenes degradation. An initial lag-time with no or very limited carotenes degradation was observed: from one week at 37 °C up to 3 months (SDE) or more (FDE) at 21 °C. The activation energies (Ea) varied between 66.6 and 79.5 kJ/mol, and Ea values tended to be higher in FDE than in SDE. Carotenoids are widely distributed fat-soluble pigments, which naturally exhibit the red, orange, and yellow color of fruits and vegetables. Recent interest in carotenoids mostly arises from their health benefits. Some carotenoids serve as precursors of vitamin A. Vitamin A is essential for vision, reproduction, and bone health . In addiIncreasing consumer awareness and legislative actions against the use of synthetic colorants pushed the food industry to create functional food products with natural pigments, such as carotenoids. However, food industry expansion leads to the production of a large amount of waste materials rich in valuable molecules, whose conversion into economically valuable goods is supported within the circular food processing concept. Carrot is among the 10 most important vegetable crops in the world. About 25–30% of globally produced carrots are aesthetically unsuitable for the fresh market and are transformed into juices, discarding 30–40% of raw material as exhausted pulp [The extraction of carotenoids with edible oils has been extensively studied ,7. This Besides poor bioavailability from native sources, carotenoids are very susceptible to degradation. In fact, the high number of conjugated double bonds makes carotenoids prone to autooxidation and isomerization during food processing and storage . A promiSome information on carotenoid oil extraction combined with encapsulation exists ,8,15, buThe carotenoids found in the encapsulates were α-carotene, β-carotene, and cis-β-carotene. The initial carotenes content corresponded, respectively, to 5.5, 18.1, and 5.8 mg/kg DM for the spray-dried encapsulate (SDE) and to 5.5, 18.5 and 5.0 mg/kg DM for the freeze-dried encapsulate (FDE). The cis-β-carotenes likely derive from isomerization reactions of all-trans-β-carotene and are generally absent in fresh carrots  or very 0 − kt, where C0 is the initial concentration, k is the reaction rate constant, t is the reaction time (days). The degradation rate depended on the temperature, but at 4 °C the carotenes were stable for at least 413 days. The cis-β-carotene exhibited an atypical pattern increasing at first, likely because of isomerization side reactions, and then decreasing for its degradation to epoxides, apocarotenones, and apocarotenals [rotenals . This berotenals .The faster oxidation of the superficial layer, followed by delayed depletion of encapsulated carotenoids, likely induces a pseudo-first order kinetic of carotenoids degradation in dried preparations . In factAdditionally, they fall within the range (5.5–87.2 weeks) of trans-β-carotene encapsulated in maltodextrins, variously added with glucose, galactose, or lactose, and stored at 25 °C . Similarp ≤ 0.05) than spray-drying against carotenes degradation, such as for α- and β-carotene at 30 and 21.3 °C. An initial lag-time with no or very limited carotenes degradation was observed, ranging from 1 week at 37 °C up to 3 months (SDE) or more (FDE) at 21 °C. The kinetics at 4 °C for all compounds and the kinetics of cis-β-carotene at all temperatures had low regression coefficients suggesting to exclude them from further analysis. Comparing the slopes of the regression lines of FDE and SDE at the same temperature , the t-tThe Arrhenius plot  illustra1/2 and Ea depend on the source of carotenoids, the core material, and the storage conditions. During storage the carotenoids can form complex structures with a matrix which influence Ea. Since the FDE samples were found to be more stable, slower release of carotenoids, higher Ea and z values of both carotenes are reasonable; additionally, the concentration of carotenoids in encapsulates is also an important factor [Our values on average were higher than the Ea observed in carotenes (range: 23.01–73.35 kJ/mol) by several Authors ,35,36,37t factor . n-hexane (97% HPLC grade), tetrahydrofuran ; ethyl acetate ; ethanol (96%), methanol (HPLC grade), dichlorometane ; butylated hydroxytoluene .The chemicals used were β-carotene standard ; pyrogallol ; sodium chloride, potassium hydroxide ; The carrot waste was obtained from the “Nectar” beverage industry . The waste was immediately packed, freeze-dried, and stored at −20 °C until use. The sunflower oil used for carotenoids extraction, purchased from a local supermarket, was produced by the edible oil manufacturing company “Dijamant” . The whey protein concentrate was purchased from Olimp Laboratories . The inulin was provided by Elephant Pharma .w/v) at 25 °C by stirring with a B800E high-speed blender for 30 min , using time shifts of 10 min blend and 5 min pause to avoid heating. The mix was then centrifuged at 2470× g for 10 min , the supernatant was recovered and stored at 4 °C in a dark glass bottle, wrapped in foil, for further use.Freeze-dried carrot waste was mixed with sunflower oil (1:10 w/v) ratio and stirred until the temperature reached 30 °C; for spray-drying, the wall material was mixed with distilled water in a 1:8 (w/v) ratio following the same procedure. The wall material-oil extract solutions (100 g/60 mL), with the addition of the emulsifier Tween 80, were homogenized at 11,000 rpm by an Ultra-Turrax® T25  for 3 min at room temperature. The first formulation was kept overnight at −80 °C and then freeze-dried at −40 °C for 48 h with a Christ Alpha 2–4 LSC , to ensure complete drying; the freeze-dried encapsulates (FDE) were stored at −20 °C until further use. The second formulation was spray-dried using a Büchi mini B-290  at an inlet temperature of 130 °C and an outlet temperature of 65 ± 2 °C. The spraying air flow and liquid feed rate were 600 L/h and 8 mL/min, respectively. The spray-dried encapsulates (SDE) were stored at −20 °C until further use.The enriched oil was encapsulated by two different techniques, i.e., freeze-drying and spray-drying, according to the optimal conditions determined in a preliminary study . The optThe encapsulates were placed in two half-filled 50 mL screw-capped tubes per sample, stored in the dark at different temperatures: +4 °C, +21.3 °C , +30 °C , +37 °C  and analysed for their carotenoids content after different storage times up to 413, 413, 200, and 143 days, respectively.v/v). The organic layer was collected and evaporated under vacuum, followed by nitrogen drying; the residue was dissolved in 2 mL methanol:dichlorometane (1:1 v/v) and filtered through a 0.45 μm PTFE membrane. The filtered solution (20 μL) was injected in a HPLC system including: column Vydac 201TP54 C18, 250 × 4.6 mm, 5 μm ; LiCHrospher WP 300 RP-18, 5 μm ; mobile phase, methanol:tetrahydrofuran stabilized with 0.1% butylated hydroxytoluene ; flow rate, 1 mL/min; pump Waters 510 . Carotenoids were detected at 445 nm, using a Waters 996 series photodiode array detector , controlled by the software Millenium 32 Chromatography Manager . The wavelength range was 200–600 nm. The quantification of the peaks was performed with the external standard method by preparing the calibration curve of β-carotene for all the compounds. The results are expressed in mg/kg dry matter (DM).Carotenoids quantification was performed by reverse phase HPLC . BrieflyC is the concentration of the compound (mg/kg DM), k is the reaction rate constant, t is the reaction time (days) and n is the order of the reaction [t1/2) [time for the concentration of a reactant to fall to half its initial value; C0 is the initial concentration, and Ea is the activation energy of the reaction (kJ/mol), A is the pre-exponential factor, R is the gas constant (8.314 J/mol·K) and T is the absolute temperature (K). The z value To determine the degradation reaction order of each carotenoid, both zero- and first-order kinetics were hypothesized by applying the general reaction rate expression reaction . The ord® Centurion XVI v16.2.04 statistical software .The average values, standard deviations, linear regression, and kinetic constants were computed using the Microsoft Excel 2016 software . The comparison between the slopes of the regression lines was performed with the STATGRAPHICSIn encapsulated carrot waste extracts, the carotenoids degradation rate was dependent on the temperature, following a zero-order kinetics, while at 4 °C the carotenes were stable for at least 413 days. The freeze-drying technique was more effective against the degradation of α- and β-carotene, particularly at 30 and 21.3 °C. An initial lag-time with no or very limited carotenes degradation was noticed and spanned from one week at 37 °C up to 3 months (SDE) or more (FDE) at 21 °C. These findings suggest that encapsulation of carrot waste extract by freeze-drying and spray-drying, using as wall materials whey protein and whey protein-inulin blend, respectively, ensures good carotenes protection during storage. In conclusion, the encapsulates of carrot waste oil extract showed a good shelf life even at room temperature, suggesting their use as a promising ingredient for the preparation of food products. Further studies might be needed to evaluate and optimize their shelf life performance under different packaging materials and conditions.


Induction of ferroptosis has shown promising antitumor effects in ovarian cancer cells, but the existence of still undefined genetic and metabolic determinants of susceptibility has so far limited the application of ferroptosis inducers Erastin and/or the iron compound ferlixit were used to trigger ferroptosis in HEY, COV318, PEO4, and A2780CP ovarian cancer cell lines. Cell viability and cell death were measured by MTT and PI flow cytometry assay, respectively. The “ballooning” phenotype was tested as ferroptosis specific morphological feature. Mitochondrial dysfunction was evaluated based on ultrastructural changes, mitochondrial ROS, and mitochondrial membrane polarization. Lipid peroxidation was tested through both C11-BODIPY and malondialdehyde assays. VDAC2 and GPX4 protein levels were quantified as additional putative indicators of mitochondrial dysfunction or lipid peroxidation, respectively. The effect of erastin/ferlixit treatments on iron metabolism was analyzed by measuring intracellular labile iron pool and ROS. FtH and NCOA4 were measured as biomarkers of ferritinophagy.Here, we provide evidence that erastin is unable to induce ferroptosis in a series of ovarian cancer cell lines. In HEY cells, provided with a high intracellular labile iron pool, erastin treatment is accompanied by NCOA4-mediated ferritinophagy and mitochondrial dysfunction, thus triggering ferroptosis. In agreement, iron chelation counteracts erastin-induced ferroptosis in these cells. COV318 cells, with low baseline intracellular labile iron pool, appear resistant to erastin treatment. Notably, the use of ferlixit sensitizes COV318 cells to erastin through a NCOA4-independent intracellular iron accumulation and mitochondrial dysfunction. Ferlixit alone mimics erastin effects and promotes ferroptosis in HEY cells.This study proposes both the baseline and the induced intracellular free iron level as a significant determinant of ferroptosis sensitivity and discusses the potential use of ferlixit in combination with erastin to overcome ferroptosis chemoresistance in ovarian cancer. During e models . The PARe models . Howevere models . This phe models . Hence, KRAS mutation is the first genetic determinant found to sensitize cancer cells to erastin-induced ferroptosis (KRAS-mutated cells may show high levels of the transferrin receptor 1 (TFR1) and decreased expression of the ferritin heavy chain (FtH), thus leading to enhanced iron intake, inhibited iron storage, and accumulation of the labile iron pool (LIP)  . As experoptosis . Similarroptosis . Converssis . Rather, we demonstrate that sufficient constitutive and treatment-induced amount of LIP is mandatory for erastin-mediated ferroptosis and that the use of ferlixit, an iron compound normally used to treat patients suffering anemia, overcomes erastin-mediated ferroptosis resistance. Overall, in this study, we validate the concept of “iron addiction” as a determinant of ferroptosis susceptibility in OVCA cells and we propose the combined use of ferlixit and erastin as a new therapeutic approach.Here, we show that erastin efficacy in OVCA cells might be independent of 2 atmosphere. The cisplatin-resistant ovarian cancer cell lines (PEO4 and A2780CP) were obtained from the American Type Culture Collection . These cells were cultured in RPMI 1640 , supplemented with 10% (v/v) fetal bovine serum (FBS) , L-glutamine and 1% (v/v) penicillin and streptomycin  in adherent cultures at 37°C in a humidified incubator with 5% CO2 atmosphere. To maintain the resistance, 1 µM cisplatin was added to the medium of A2780CP and PEO4 cells every two to three passages. All cell lines were tested for mycoplasma contaminations and STR profiled for authentication.The human epithelial ovarian cancer cell lines HEY and COV318 were purchased from the American Type Culture Collection . COV318 and HEY cells were grown in DMEM medium  supplemented with 10% (v/v) fetal bovine serum (FBS) , L-glutamine (0.3 g/l) and 1% (v/v) penicillin and streptomycin  at 37°C in a humidified incubator with 5% COFerlixit  and cisplatin  were obtained from the outpatient pharmacy at the Unit of Gynecologic Oncology, Magna Graecia University of Catanzaro; deferoxamine mesylate salt (DFO), erastin, ferrostatin-1 (Fer-1), hemin and cloroquine (Cq) were purchased from Sigma Aldrich ; the antioxidant (±)-6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid (trolox) was ordered from Cayman Chemical . Cells were seeded in a 6-well plate in serum-free medium. Each compound was used at the following final concentrations: ferlixit at 100 µM and 250 µM for 1, 8, 24 or 48 h; cisplatin at 150 μM for 24 h; DFO at 200 µM for 24 h; erastin at 8 µM and 25 µM for 1, 8 or 24 h; Fer-1, hemin and Cq at 10 µM for 8, 24 or 48 h; and trolox at 200 μM for 8 h. Treatments were performed at least three times on independent biological replicates.4 cells/well were seeded into a 96-well flat-bottom plate and were subjected to drug treatment. There were quintuplicates for each cell type. Fresh MTT , re-suspended in phosphate-buffered saline (PBS) was added to each well for a final concentration of 0.5 mg/ml. After 4 h incubation, culture medium was discarded and replaced with 200 μl of isopropanol to solubilize formazan crystals. Optical density was measured at 595 nm in a spectrophotometer. Each experiment was performed in triplicate.3--2,5-diphenyltetrazolium bromide (MTT)  assay was performed to detect proliferation of HEY and COV318 cells untreated or treated with erastin, cisplatin and ferlixit. A total of 10Growth rate of HEY and COV318 cells was obtained using the trypan blue dye exclusion method. Cells were counted after 24, 48, and 7h. Each experiment was performed in triplicate.6 cells/well were seeded in 6-well plates overnight followed by the various treatment. Cells were centrifuged and the relative pellets were incubated with PI staining in the dark at 37°C for 15 min. Samples were then washed twice with PBS. The cellular fluorescence was analyzed by FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A total of 2 × 104 event was acquired for each sample from three independent experiments. Fluorescence was measured using FlowJo software program . Each experiment was performed in triplicate.Approximately 1 × 105 cells/well and grown overnight. Following treatment with erastin, ferlixit, hemin, and DFO, morphological changes of “ballooning” phenotype were observed by light microscopy. Images were captured with Leica DM IL LED inverted phase contrast microscope .Cells were seeded in 6-well plates at density of 4 × 106 cells/well) were plated in 100 mm culture dishes. Cells were then treated with erastin, DFO and/or ferlixit. Cells were centrifuged and the relative pellet were fixed for 3 h with 3% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4). After washing in PBS for 15 min, samples were post-fixed in osmium tetroxide (1%) for 2 h, dehydrated in graded acetone, and then progressively embedded in acetone/resin with final embedment in pure resin . Ultrathin sections (60–90 nm in thickness) were cut with a diamond knife, collected on copper grids (G300 Cu), and then examined with a Jeol JEM 1400‐Plus electron microscope operating at 80 kV.HEY and COV318 cells , according to the instructions of the manufacturer. CM-H2DCFDA fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences) and data were processed with FlowJo software . Each experiment was performed in triplicate.5 cells/well and grown overnight. After treatments, cells were loaded with 2.5 μM BODIPY™ 581/591 C11 for 30 min at 37°C. After 30 min of loading, unincorporated dye was removed by washings twice with PBS. Samples were then centrifuged at 1,000 r.p.m. for 3 min and the pellets were resuspended in 500 μl of PBS. The cell suspension was subjected to the flow cytometry analysis to analyze the amount of lipid ROS within cells. Oxidation of BODIPY-C11 resulted in a shift of the fluorescence emission peak from ~590 to ~510 nm proportional to lipid ROS generation . Briefly, cells were seeded in 6-well plates at a density of 4 × 10neration . The flu5 cells per plate) and treated with erastin or ferlixit. Cells were washed with ice-cold PBS and homogenized on ice in 300 μl of the MDA lysis buffer with 3 μl BHT (100×), then centrifuged  to remove insoluble material. Approximately 200 μl of the supernatant from each homogenized sample were placed into a microcentrifuge tube; then 600 μl of the thiobarbituric acid (TBA) solution were added into each vial to form the MDA-TBA adduct. The mixture was incubated for 1 h at 95°C followed by ice-cooling for 10 min. Lipid peroxidation was determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm)/fluorometric adduct, proportional to the MDA present. The absorbance was measured in a spectrophotometer. Each experiment was performed in triplicate.Cellular MDA levels were determined by using the lipid peroxidation assay kit  according to the protocol of the manufacturer. Briefly, cells were seeded in a 6-well plate . After treatments, cells were incubated with 5 µM MitoSOX Red for 10 min at 37°C and then analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A minimum of 20,000 cells was analyzed per condition. Fluorescence was measured using FlowJo software program . Each experiment was performed in triplicate.Changes in the mitochondrial membrane potential were analyzed by staining the cells with TMRE (tetramethylrhodamine ethyl ester) dye . Briefly, cells were cultured in 6-well plates and, upon treatments, were incubated with 100 nM TMRE dye for 30 min at 37°C and then washed with PBS. Samples were then centrifuged at 1,000 r.p.m. for 3 min and the pellets were resuspended in 500 μl of PBS. TMRE fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A minimum of 20,000 cells was analyzed per condition. Fluorescence was measured using FlowJo software program . Each experiment was performed in triplicate.5 cells/well and grown overnight. Then cells were loaded with 0.25 μM calcein acetoxymethyl ester   for 30 min at 37°C. After washing twice with PBS, cells were treated with 200 μM 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone or L1)  or left untreated. Following staining, and washing PBS, cells were analyzed by a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The difference in the mean fluorescence index between chelator-treated and untreated cells  reflects the amount of LIP. Each experiment was performed in triplicate.The intracellular labile iron pool (LIP) was quantified by monitoring the recovering of calcein fluorescence induced by iron chelators after the calcein fluorescence was quenched by intracellular LIP. Briefly, cancer cells were seeded in 6-well plates at a density of 4 × 10g for 30 min at 4°C to eliminate insoluble fragments. For nuclear and cytoplasmic protein extraction, 2 × 107 cells were washed once with PBS (1×) and cell pellets were resuspended in 200 μl of Lysis Buffer w/NP40. After 5 min incubation in ice, all tubes were centrifuged at 3,500 r.p.m. for 5 min at 4°C. The supernatant, containing cytoplasmic protein fraction, was transferred in a new eppendorf tube, while the pellet, containing cell nuclei, was washed in PBS (1×). To obtain nuclear protein content, the latter was resuspended in 1 ml of Lysis Buffer w/o NP40 and centrifuged twice at 3,500 r.p.m. at 4°C for 5 and 10 min, respectively. After these two centrifugations, the pellet was resuspended in 300 μl of Lysis Buffer w/o NP40 and 300 μl of Cushing Buffer were added. This mix solution was centrifuged at 6,000 r.p.m. for 10 min at 4°C and then resuspended in 300 μl of Resuspension Buffer. Three freeze-thaw cycles were performed according to protocol. Following a last centrifuge at 9,500 r.p.m. for 15 min at 4°C, the supernatant, containing nuclear proteins was collected. For recipes of buffers used for selective nuclear and cytoplasmic protein extraction refer to Chirillo et al.  and total cell lysates were prepared using RIPA buffer containing 1 M Tris HCl, Triton X-100, 3 M NaCl, 0.5 M EDTA, 10% SDS supplemented with cOmplete™ Protease Inhibitor Cocktail provided in EASYpacks  as described by Biamonte et al. . Brieflyo et al. .via using the ECL western blotting detection system  and acquired by Uvitec Alliance Mini HD9 . To ensure equal loading of proteins a goat polyclonal anti-γ-Tubulin (γ-TUB) antibody  was used. The protein band intensity on western blots was quantified and normalized to that of γ-TUB by using ImageJ software (http://rsb.info.nih.gov/ij/).Protein amount was determined using the Protein Assay Dye Reagent Concentrate . Each protein sample (40–50 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes . After blocking with 5% milk, incubation with primary antibody was performed overnight at 4°C. Antibodies against FtH , NCOA4  were purchased from Santa Cruz Biotechnology ; antibodies against VDAC2  and GPX4  were purchased from Abcam . After incubation with peroxidase-conjugated secondary antibodies  for 1 h at room temperature, the signals were visualized p53 and BAX were calculated by the 2−ΔΔCt method and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Each experiment was performed in triplicate.Total RNA was extracted using the Trizol method  according to the instructions of the manufacturer , 26. Thet-test. We were interested in determining whether the means of more than two groups were equal or not, thus, we performed an analysis of variance (ANOVA). A p-value <0.05 was considered statistically significant.Overall data are represented as mean ± standard deviation (SD) of at least three biological replicates. When appropriate, data were analyzed by performing a simple comparison between two groups using Student’s bona fide ferroptosis inducer in OVCA cell lines. Taking into account that erastin was originally selected as an agent able to trigger ferroptosis exclusively in RAS-mutated cancer cells, we selected four OVCA cell lines, three of which are KRASwt  and one is KRASG12D (HEY). Furthermore, this panel includes both cisplatin-sensitive (HEY and COV318) and cisplatin-resistant (PEO4 and A2780CP) cells (see cell viability assay in p <0.05) and further broke down with 25 μM erastin for 8 h . Conversely, COV318 and PEO4 cells are insensitive to both erastin concentrations, with cell viability nearly of 90% upon treatments. Similar results were obtained by using propidium iodide (PI) staining. Flow cytometry analysis highlighted a significant increase in cell death in HEY and A2780CP cells treated with both 8 μM (25.1% PI+ HEY and 29.3% PI+ A2780CP cells) and 25 μM erastin (91.8% PI+ HEY and 59.1% PI+ A2780CP cells) for 8 h .Then, we investigated the molecular basis underlying erastin-induced ferroptosis in HEY cells. Erastin treatment may alter mitochondrial membrane potential (ΔΨ), thus ultimately leading to mitochondrial dysfunction . Hence, DFO ΔMFI:2315 vs HEYuntreated ΔMFI:4780), as well as both total and mitoROS . Albeit leading to a slight mitochondrial membrane depolarization (HEYDFO TMRM MFI:1501 vs HEYuntreated TMRM MFI:2152) and reduction of VDAC2 protein levels, DFO alone did not affect mitochondrial ultrastructure or HEY cell death (3.7% PI+ HEYDFO vs 2.02% PI+ HEYuntreated)      TMRM MFI:6294; HEYerastin (8 h) TMRM MFI:1195; HEYDFO/erastin (1 h) TMRM MFI:1541; HEYDFO/erastin (8 h) TMRM MFI:1143)  (200 μM) for 24 h; then, culture medium was replaced, and cells were treated with erastin (8 μM for 8 h). First, DFO treatment alone significantly reduced the LIP  and that the combination (100 μM for 24 h) with erastin (8 μM for 8 h) broke down cell viability to less than 2% (p <0.05)     . Plus, the lowest dose of ferlixit (100 μM) was used as pre-treatement for 24 h followed by administration of 8 μM erastin (8 h). We observed that the sole administration of ferlixit at 250 μM for 48 h reduced COV318 cell viability to ~45% (p <0.05) and the relative PI+ HEY cells were 50.2%. Notably, at 100 and 250 μM for 48 h, ferlixit caused massive cytotoxic effects (PI+ HEY100 μM ferlixit (48 h): 97.5%; PI+ HEY250 μM ferlixit (48 h): 95.4%) (untreated CM-H2DCFDA:24046 vs HEYferlixit CM-H2DCFDA:63302) (HEYuntreated MitoSOX Red:261 vs HEYferlixit MitoSOX Red:1104)  and we found that already at the lowest concentration cell viability decreased to ~40% (+ HEY) while treatment with 10 μM for 48 h moderately promoted cell death in COV318 cells (34.6% PI+ COV318). Therefore, we combined hemin (10 μM for 48 h) with erastin (8 μM for 8 h) and we observed a significant impact on cell death also in COV318 (94.7% PI+ COV318). The analysis of the “ballooning phenotype” further supported the role of hemin in ferroptotic cell death  normally used to treat anemia, significantly sensitizes OVCA cells to ferroptosis either alone or in combination with erastin. Ferlixit-induced ferroptotic cell death was confirmed by the use of the specific ferroptosis inhibitor Fer-1 (p53 and BAX expression levels and ii) the use of autophagy inhibitor (Cq) did not interfere with the cytotoxic effect of the two drugs alone or used as combined treatment (Notably, we demonstrate that the use of ferlixit, an iron compound (Fereatment , 39. In reatment , no incrreatment , 59. Futin vivo studies. Taking into account that, so far, the use of FINs in vivo has been limited by unsatisfactory pharmacokinetic and physicochemical properties (Overall, our findings show that both the baseline and the treatment-induced LIP content determines the response of OVCA cells to erastin-mediated ferroptosis. In particular, ferroptosis occurs when LIP reaches a threshold level causing a detrimental oxidative stress. Furthermore, we demonstrate the administration of low doses of ferlixit improves the effect of erastin-based antitumor approach and, thus, put the ground for the use of this combined therapy in future operties , the ideThe original contributions presented in the study are included in the article/FC and FB designed this study. AMB, AS, and MS performed the analysis. IDP performed electron microscopy analysis. FC and FB wrote the manuscript. AMB, MCF, DT, SL, FC, and FB reviewed the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nardostachys jatamansi DC. was an important plant used to process the traditional Chinese herbal medicines “gansong”. Pancreatic cancer was the fourth most common cause of cancer-related death in the world. Hence, there was an urgent need to develop novel agents for the treatment of pancreatic cancer. In this paper, nardoguaianone L (G-6) is isolated from N. jatamansi, which inhibited SW1990 cells colony formation and cell migration, and induced cell apoptosis. Furthermore, we analyzed the differential expression proteins after treatment with G-6 in SW1990 cells by using iTRAQ/TMT-based quantitative proteomics technology, and the results showed that G-6 regulated 143 proteins’ differential expression by GO annotation, including biological process, cellular component, and molecular function. Meanwhile, KEGG enrichment found that with Human T-cell leukemia virus, one infection was the most highly enhanced pathway. Furthermore, the MET/PTEN/TGF-β pathway was identified as a significant pathway that had important biological functions, including cell migration and motility by PPI network analysis in SW1990 cells. Taken together, our study found that G-6 is a potential anti-pancreatic cancer agent with regulation of MET/PTEN/TGF-β pathway.Natural products (NPs) were a rich source of diverse bioactive molecules. Most anti-tumor agents were built on natural scaffolds.  Nardostachys jatamansi DC. could be one important resource for the discovery of bioactive natural products [N. jatamansi have yet to be identified.Various traditional Chinese medicines and their associated active compounds have been remarked to exhibit powerful anti-cancer activities and important health benefits, including plant origin compounds such as alkaloids, terpenoids, flavonoids, plant polysaccharides, polyphenols, organic acids and lignans ,2,3. Curproducts , includiproducts , antioxiproducts , anti-tuproducts , anti-inproducts , anti-Paproducts , and oxiproducts . HoweverN. jatamansi, tested their inhibition on human pancreatic cancer cell lines growth [N. jatamansi as a compound with original structure and a set of interesting biological properties [We extracted and isolated novel compounds from roots and rhizomes of s growth , and fouoperties . We dete50 value was 2.1 ± 0.3 μM; it’s similar to that of GEM (1.8 ± 0.5 μM). Furthermore, in order to study the G-6’s inhibition performance towards SW1990 cells’ proliferation, SW1990 cells were analyzed using a cell clone formation trial. The constructed cell colonies were numbered after 10 days. Compared with control, G-6 decreased number of cell colonies with 1 μM and 10 μM, and GEM as a positive control group  are involved in extracellular matrix degradation and can both activate latent TGF-β . Therefohttps://www.omicshare.com/tools/, accessed on 10 July 2022). Using a clustermap generated from data analysis , we further show a similar pattern by plotting a heatmap for each event  is community-based bioinformatics resource that supplies information about gene product function using ontologies to represent biological knowledge. To understand proteomic change during follicular development and maturation among the above two groups, we screened reliable proteins and differential proteins. A total of 9864 reliable proteins were found in this study. The number of significant differential abundance was also a statistic and displayed by the positive and negative column figure A. For inThe differentially expressed proteins in molecular function (MF), cell component (CC), and biological process (BP) and categories by GO study, accordingly, was illustrated in p-value and the number of proteins enriched to this pathway, with the 10 pathways showing the most significant enrichment. Among them, Human T-cell leukemia virus 1 infection was the most significantly enriched pathway. The PPI network analysis found that some DEPs interact with each other, such as MET/PTEN/TGF-β. These key focus hubs expressed a crucial biological activities biological adhesion, cell migration, enzyme activity and biological regulation . Other reagents materials including Dulbecco’s Modified Eagle Medium , MTT -2,5-diphenyl-2-H-tetrazolium bromide, Kum-amoto, Japan), DMSO (Dimethyl sulfoxide) . The primary antibodies MMP2/9   and FITC-conjugated Goat Anti-Rabbit IgG (H + L) Secondary Antibody .Nardoguaianone L (G-6) was obtained by our research group, and the HPLC purity >98% . For in v/v) fetal bovine serum  in a humidified atmosphere contained 5% CO2 at 37 °C.SW1990 cells  were cultured in DMEM that contained 100 μg/mL streptomycin, 100 U/mL penicillin  and 10% (2 atmosphere. Cell viability assessed by MTT assay [570 of treated group − OD570 of blank group)/(OD570 of untreated group − OD570 of blank group) × 100%.Cells were incubated at 37 °C in a 5% COTT assay . G-6 andSW1990 cells plated on coverslips were either treated or untreated using different levels of compound G-6. After 24 h, the cells were permeabilized in 2% bovine serum albumin and 0.1% Triton X-100 and fixed with 4% paraformaldehyde as illustrated in previous work . The sam5) were grown on glass slides to 70–80% confluency, and were then treated with 20 µM and 40 µM of compound G-6 for 24 h at 37 °C in an atmosphere containing 5% CO2. Following treatment, the cells were fixed, washed twice with phosphate-buffered saline (PBS) and stained with Hoechst 33258  staining solution, according to the manufacturer’s protocol [For the morphological investigation, SW1990 cells (1 × 10protocol . Morphol5 cells/mL) were plated in 6-well plates and then treated with vehical, 20 μM and 40 μM of compound G-6 for 24 h. The cells apoptosis assay according to protocol of apoptosis kit  [SW1990 cells  .SW1990 cells were synchronized in a low serum medium for 8 h, and the cell monolayers were then damaged and created a linear 2 mm width wound. Different concentrations of G-6 were added and the cells were incubated for 0 h, 10 h, 24 h and 48 h. Images were received using a microscope  and the data are declared as the percentage inhibition of migration rate compared with untreated cells .g for 10 min, the supernatants were collected, and protein concentrations were determined by Pierce™ BCA Protein Assay Kit. And more steps are referred to Burke et al. [Lysis solution  and Extraction Buffer  were added to each sample , and thee et al. .Ten-plex TMT labeling was performed according to the manufacturer’s instructions . Equilibrate the TMT Label Reagents to room temperature and added 20 μL of anhydrous acetonitrile into each tube with intermittent shaking for 5 min, and centrifuge the tube to gather the solution. Following, TMT Reagent and alkylated protein was mixed and incubated the reaction for 1 h at room temperature. The reaction was quenched when added 1 μL of 5% hydroxylamine to the sample and incubated for 15 min. Finally, Vortex each tube to mix, combine samples at equal amounts in new microcentrifuge tube and stored at −80 °C.g for 2 min to remove the solution and pack the resin material. Discard the liquid. Remove the top screw cap and load 300 μL of ACN into the column. Replace the cap, place the spin column back into a 2.0 mL sample tube and centrifuge at 5000× g for 2 min. Discard ACN and repeat wash step. Wash the spin column twice with 0.1% TFA solution. The column is now conditioned and ready for use. Dissolve the digested sample in 300 μL of 0.1% TFA solution. Place the spin column into a new 2.0 mL sample tube. Load 300 μL of the sample solution onto the column, replace the top cap and centrifuge at 3000× g for 2 min. Retain eluate as “flow-through” fraction. Place the column into a new 2.0 mL sample tube. Load 300 μL of 5% ACN, 0.1% TEA to remove unreacted TMT reagent. Place the column into a new 2.0 mL sample tube. Load 300 μL of the appropriate elution solution and centrifuge at 3000× g for 2 min to collect the fraction. Repeat for the remaining step gradient fractions using the appropriate elution solutions in new 2.0 mL sample tubes. Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation.Prepare solutions in 1.5 mL tubes according to requirement. Remove the protective white tip from the bottom of the column and discard. Place the column into a 2.0 mL sample tube. Centrifuge at 5000× m/z 350–1500 with a mass resolution of 60,000 (at m/z 200).Samples were resuspended with Nano-UPLC buffer A (0.1% FA) and buffer B 20% 0.1% formic acid in water-80% acetonitrile. The online Nano-UPLC was employed on the Easy-nLC 1200 System . The samples were washed by Nano-UPLC mobile phase at 600 nL/min. An elution gradient of 4–10% acetonitrile (0.1% formic acid) in 5 min, from 10% to 22% B for 80 min, from 22% to 40% B for 25 min, from 40% to 95% B for 5 min and from 95% to 95% B for 5 min. The gradient was used on an analytical column . Data attainment was carried out using a Orbitrap Eclipse  fitted with a Nanospray. The Orbitrap Eclipse instrument was performed with a data-dependent top-20 experiment with 60K resolution for the full MS scans, 30 K resolution for high energy collisional dissociation (HCD) MS/MS scans. A top 20 method was used. Full MS scans were received in the Orbitrap mass analyzer over the range ® search engine, constrained with a precursor mass tolerance of 20 ppm and fragment mass tolerance of 20 ppm [p < 0.05).The collected spectral result files were studied via Proteome Discoverer™ 2.5 software by the SEQUESTf 20 ppm . Carbamihttp://www.omicsbean.cn, accessed on 10 July 2022) on online databases.Gene ontology (GO) and KEGG pathway analysis of the ensemble of detected proteins was performed through OmicsBean  and compared by Dunn’s Multiple Comparisons Test. N. jatamansi involves multiple mechanisms that culminate in an overall inhibition of tumor growth through both inhibition of proliferation and induction of apoptosis, also including inhibition of tumor cell migration. These results may be explained by the fact that G-6 regulated key factor in quantitative proteomics levels. Therefore, G-6 inhibited the MET/PTEN/TGF-β pathway, mediating these currents contributions to the anti-tumor activity, and showed that G-6 is a promising anti-pancreatic cancer agent candidate potently in vitro. G-6 may act as a new structural framework for the subsequent development of anti-pancreatic cancer agents.In conclusion, G-6 from


PALB2. Methods: Our work is based on the exposure of MCF10A mammary epithelial cells to seven compounds of our exposome  alone or in cocktail. The qMSRE and RMS techniques were used to study the impact of these exposures on the level of methylation and mutation of the PALB2 gene. Results: Here, we have found that exposome compounds  promoting the cytosine methylation and the 5-methylcytosine deamination have the ability to promote a specific C > T mutation in the PALB2 gene. Interestingly, we also noted that the addition of exposome compounds promoting the TET-mediated conversion of 5-methylcytosine (Ascorbic acid and iron) abrogates the presence of C > T mutation in the PALB2 gene. Conclusions: Our study provides a proof of concept supporting the idea that exposomes can generate genetic mutation by affecting DNA methylation/demethylation.Background: Deregulation of DNA methylation/demethylation reactions may be the source of C > T mutation via active deamination of 5-methylcytosine to thymine. Exposome, that is to say, the totality of exposures to which an individual is subjected during their life, can deregulate these reactions. Thus, one may wonder whether the exposome can induce C > T mutations in the breast cancer-predisposing gene  DNA methylation is an epigenetic process involving the transfer of a methyl group CH3) to the 5’ position of a cytosine from S-adenyl methionine (SAM) molecules. Proteins of the DNA methyltransferase family or DNMTs are the enzymes catalyzing this transfer . In cont to the 5Literature reports several crosstalks between DNA methylation/demethylation and genetic alterations: (i) hypomethylation of retrotransposons that promotes chromosomal instability ,5,6, (iihttps://www.cancer.gov/publications/dictionaries/cancer-terms (accessed on 16 August 2022)). Today, several molecular mechanisms are proposed to be at the origin of driver C > T mutations including spontaneous and AID/APOBEC-catalyzed deamination of 5 mC and defective DNA mismatch repair [c.929C > TERBB2, c697C > TPTEN, c637C > TTP53, c.5965C > TARID1A, c.394C > TIDH1, c.4183C > TBRCA1 and c.8827C > TBRC2A. C > T mutations were also detected in the PALB2 gene with the c.1240C > T [As reported by Alexendrov et al., (2020), C > T mutation is part of the repertoire of mutational signatures in human cancer . This tyh repair . C > T m240C > T , c.2257C240C > T , c.3256C240C > T , and c.1240C > T ,18,19. The exposome, that is, the totality of exposures  to which an individual is subjected during their life, has the capacity to affect DNA methylation/demethylation reactions, and by ricochet, the presence of C > T mutations, putatively . Indeed,PALB2 gene. In our work, the PALB2 gene choice as a “demonstrator gene” is based on the fact that this gene is methylated or mutated in different types of cancer including breast cancer [In this work, we provide a proof of concept supporting the idea that different compounds of exposome have the ability to guide the DNA methylation/demethylation reactions to promote C > T mutation in the t cancer ,31,32,33PALB2 gene by enhancing or inhibiting DNA methylation/demethylation reactions  for 3 weeks to three doses of Folate equal to the recommended daily intake (RDI), three times RDI (3RDI), and 10 times RDI (10RDI) [PALB2 gene. Based on the ClinVar Miner database, we focused our study on 4 C > T mutations: c.1027C > T, c.1240C > T, c.2257C > T, and c.3256C > T. The choice of these four mutations is based on the fact that these mutations must be included in a “methylable” CG dinucleotide. In practice, the Folate-induced methylation of cytosine was then analyzed using the MeDIP method. The MeDIP assay showed that only the c.1027C > T mutation-prone region was methylated following Folate supplementation at the indicated RDI (PALB2c.1027C > T).Our first hypothesis is based on the fact that Folate could induce the methylation of cytosine giving birth to C > T mutations in the  (10RDI)   promoted PALB2c.1027C > T  assay was developed to study the presence of 027C > T A. RDI/Diuron3MAC/PFOA3MAC could induce PALB2c.1027C > T. Our data indicate that FolateRDI/Zn5RDI/PFOA3MAC and FolateRDI/Zn10RDI/PFOA3MAC cocktails promoted PALB2c.1027C > T (APOBECs enzymes being zinc (Zn)-dependent deaminases , we also027C > T B. PALB2c.1027C > T. For this purpose, we considered glyphosate, ascorbic acid, and iron since these compounds increased the TET3 expression (3RDI) and Iron at 3 RDI (Fe3RDI) limited the presence of PALB2c.1027C > T induced by FolateRDI/Diuron3MAC/PFOA3MAC or FolateRDI/Zn5RDI/PFOA3MAC  studies are an opportunity to understand the complexity of the interactions between genes and environment, and if necessary to identify predictive biomarkers for the development of pathologies such as cancer. For several years, GxE studies have benefited from the integration of complex data from epidemiological studies and omics-based technology analyses that comprehensively assess an individual’s exposome, transcriptome, epigenome, or metabolome ,42. AlthRDI/Diuron3MAC/PFOA3MAC, FolateRDI/Zn5RDI/PFOA3MAC, FolateRDI/Diuron3MAC/PFOA3MAC/Ascorbic Acid3RDI/Iron3RDI, and FolateRDI/Zn5RDI/PFOA3MAC/Ascorbic Acid3RDI/Iron3RDI exposures have the capacity to regulate the action of proteins catalyzing DNA methylation/demethylation reactions and the PALB2c.1027C > T occurrence, our work is fully in line with the deciphering of the molecular mechanisms by which the exposome is likely to modify the integrity of the genes predisposing to cancer risk  Ascorbic Acid3RDI/Iron3RDI has a “protective effect’’ against FolateRDI/Diuron3MAC/PFOA3MAC- and FolateRDI/Zinc5MAC/PFOA3MAC-induced PALB2c.1027C > T but not against FolateRDI/Diuron10MAC/PFOA10MAC- and FolateRDI/Zinc10MAC/PFOA10MAC-induced PALB2c.1027C > T (PALB2c.1027C > T since this dose (3MAC: 0.6 ng/mL) is of the same order of magnitude as that detected in the blood of different individuals by several studies  and PFOA concentration was 9.3 ng/mL with values ranging from 0.8 to 35.2 ng/mL [Our work also points out the importance of the exposure doses since (i) Folate027C > T . More ge.2 ng/mL ,51.c.1027C > T while discussing the complexity of conducting such a study in the laboratory through the consideration of the number and dose of compounds selected to mimic exposome.In conclusion, our work provides a proof of concept supporting the idea that the exposome can generate, by affecting DNA methylation/demethylation reactions, a genetic mutation predisposing to a cancer risk such as PALB2MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum , 500 ng/mL hydrocortisone , 100 ng/mL cholera toxin , 10 μg/mL insulin  and 20 ng/mL epidermal growth factor , penicillin (100 U/mL), and 2 mmol/L L-glutamine. Glyphosate  and Diuron  were purchased from Santa Cruz . DMOG (CAS No: 89464-63-1) was purchased from MedChemExpress . Ascorbic Acid , iron , Perfluorooctanoic acid , and Zinc  were purchased from Sigma . All nutraceuticals or pollutants used in our study are resuspended in PBS/DMSO5% solution.A QIAcube automate and QIAmp DNA Mini QiaCube kit  were used to isolate DNA. Qubit  was used to quantify DNA. cctaaaggtagcagtgaa and as: gcctccaaacttacagg. The mutation level was calculated using Ct values from qPCRs performed with digested and undigested DNA and the 100 × 2−ΔCt formula.Digestions were performed with adequate restriction enzymes, HpaII and AciI . Typically, 1 μg of genomic DNA was digested with 40 U of enzymes at 37 °C for 2 h in 50 μL of reaction. Control samples were treated in the same way but without the addition of the enzyme. Furthermore, 5 μL of digested and undigested mixtures were used for qPCR using QuantiFast SYBR Green PCR Kit and Rotor-Gene Q . The primers used in these qPCRs are: S: g, 5 min), aqueous supernatant was transferred to a fresh microcentrifuge tube in presence of 250 μL of chloroform. After centrifugation , aqueous supernatant was transferred to a fresh microcentrifuge tube in presence of 2 μL of the coprecipitant GlycoBlue , 20 μL 5 M NaCl and then 500 μL of 100% ethanol. The mixture was then incubated overnight in a −20 °C freezer. After centrifugation , the supernatant was removed, and the pellet was washed two times with 1 mL 70% ethanol by incubating at −20 °C for 10 min then spun again for 10 min. After discarding the supernatant using a pipette and air-drying, the pellet was resuspended in 25 μL of nuclease-free water in order to obtain the MeDIP sample. Then, 2 μL of the MeDIP sample was used for qPCR analysis. qPCR analysis was performed using QuantiFast SYBR Green PCR Kit and Rotor-Gene Q as real-time thermocycler . The primers used in these qPCR are defined in the RSM assay section. Next, the 5-methylcytosine enrichment was calculated using Ct values from qPCRs performed with DNA issue to 5 mC and IgG (a specific antibody) antibody immunoprecipitation and corresponding input sample.After treatment with 1 ng/μL RNase, 5 μg genomic DNA into 130 μL of TE buffer were sonicated with Bioruptor . Sonicated DNA was next diluted in 400 μL of TE buffer and denatured in a dry bath heating block for 10 min at 95 °C. On ice, 100 μL of cold 5 × IP and 4 μg of 5 mC antibody  were added to the denatured sonicated DNA. The mixture was then incubated on a rotating platform at 4 °C overnight. 50 μL of magnetic beads (Dynabeads M-280) were washed two times with washing buffer (1 × PBS with 0.1% BSA and 2 mM EDTA) before being resuspended in 50 μL 1x IP buffer. Then, these magnetic beads were added to the antibody-DNA mixture for incubation of 3 h on a rotating platform at 4 °C. After three washes, magnetic beads were resuspended in 250 μL Digestion Buffer and incubated in presence of 3.5 μL Proteinase K (20 mg/mL) for 2–3 h on a rotating platform at 55 °C. DNA purification was next performed by adding 250 μL phenol–chloroform–isoamyl alcohol to each tube. After centrifugation , Epigenase™5mCHydroxylase TET Activity/Inhibition Assay Kit and Epigenase™Thymine DNA glycosylase Activity/Inhibition Assay Kit , APOBEC3α, APOBEC3γ and TET3 ELISA kit  were used in accordance with the recommendations of the manufacturers.siRNA-APOBEC3γ , siRNA-APOBEC3α , siRNA-TDG , and siRNA-TET3  were transfected in MCF10A as previously described . siRNA-A , that is, a scrambled sequence devoid of specific degradation of any cellular message was used as control.t test. t. p < 0.05 was considered significant (*).All experiments were conducted at least in biological triplicates. Differences in means were assessed using the Student’s


NR1H4) plays an essential role in bile acid (BA) homeostasis. However, some novel variants and their pathogenesis have not been fully elucidated. Therefore, this research aimed to investigate the genetic characteristics of the NR1H4 gene in ICP.Intrahepatic cholestasis of pregnancy (ICP) can cause adverse pregnancy outcomes, such as spontaneous preterm delivery and stillbirth. It is a complex disease influenced by multiple factors, including genetics and the environment. Previous studies have reported that functioning nuclear receptor subfamily 1 group H member 4  than the wild-type group. Therefore, we hypothesized that these two mutations altered the protein structure of NR1H4, which impaired the function of NR1H4 itself and its target gene and caused an increase in TBA.We identified four mutations: two novel missense mutations, p.S145F and p.M185L; rs180957965 (A230S); and rs147030757 (N275N). The two novel missense mutations were absent in 1029 controls and three databases, including the 1000 Genomes Project (1000G_ALL), Exome Aggregation Consortium (ExAC) and ChinaMAP. Two web-available tools, SIFT and PolyPhen2, predicted that these mutations are harmful to the function of the protein. Moreover, compared to the wild-type protein structure, the NR1H4 mutations.To our knowledge, this is the first study to identify the novel p.S145F and p.M185L mutations in 197 ICP patients. Our present study provides new insights into the genetic architecture of ICP involving the two novel The online version contains supplementary material available at 10.1186/s12920-022-01240-w. Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease characterized by skin pruritus and abnormal liver function, such as elevated liver enzymes and increased serum TBA ≥ 10 μmol/L), that appears in the second and third trimesters of pregnancy 0 μmol/L,. The incNR1H4, ATP Binding Cassette Subfamily B Member 4 (ABCB4), ATP Binding Cassette Subfamily B Member 11 (ABCB11) and ATP Binding Cassette Subfamily C Member 2 (ABCC2), have been reported. Moreover, multiple previous studies have identified genetic variants of the NR1H4, ABCB4, ABCB11 and ABCC2 genes that contribute to the development of ICP [Obviously, ICP is a complex disease that depends on multiple factors, including genetic background, metabolites of progesterone, oestrogens, seasons and environmental background , 10, 11.t of ICP –20. AmonNR1H4 is a BA-activated transporter factor that is responsible for BA homeostasis and acts by binding to DNA response elements through the NR1H4 DNA binding domain (DBD) in the promoter of target genes , thereby activating their transcription [NR1H4 has a highly conserved ligand binding domain (LBD), which determines the specificity of NR1H4 ligands. These ligands include farnesoid derivative, BA, unsaturated fat, hepatocyte factor-1 and steroid compound [NR1H4 has four different isoforms: α1, α2, α3 and α4. The first two isoforms, which are expressed in the human liver, have a different N-terminus than the other two isoforms [NR1H4 regulates bile flow by directly inducing gene expression  to stimulate hepatic bile export [NR1H4 represses the expression of bile acid import (NTCP) [CYP7A1 and CYP8B1) [−/− transgenic mice exhibited BA pool sizes [NR1H4 maintained a stable TBA level in hepatocytes by regulating TBA synthesis, transport, secretion and metabolism.NR1H4 is both a key modulator of hepatocyte-protective pathways and a therapeutic target for cholestatic liver disease . NR1H4 icription –24. Morecompound , 26. NR1isoforms , 28. In e export , 30. Cont (NTCP)  and key  CYP8B1)  in the b CYP8B1)  in the l CYP8B1)  in the iol sizes . TherefoNR1H4 mutations resulted in altered BA levels, we hypothesized that NR1H4 mutations might also exist in ICP samples. Here, we recruited a total of 197 Han Chinese women with ICP and analysed the entire coding region of the NR1H4 gene. A total of 4 mutations, including two novel missense mutations in NR1H4, were identified in our ICP samples for the first time.Considering that women with ICP exhibited elevated serum BAs and We recruited 197 patients diagnosed with ICP disease based on clinical symptoms (skin pruritus) and laboratory investigations  between 2018 and 2020. Peripheral blood samples from 197 patients with ICP disease were collected from the Department of Obstetrics, Jiangxi Provincial Maternal and Child Health Hospital in Nanchang, China. In addition, we recorded a total of twenty-nine available clinical characteristics, which included age, body mass index (BMI), gestational weeks at diagnosis, gravidity and parity; the level of ion concentration covering K, Na, Cl, Ca, Mg and P; the counts of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), and red blood cell distribution width. SD (RDW-SD); the level of serum biochemical indices including TBA, aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IDBIL), total cholesterol (CHOL), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), uric acid (UA); and the outcomes of pregnant women and newborn babies, including birth weight, bleeding count and Apgar score. The clinical features were determined as described previously . BrieflySummary statistics for all the above clinical features investigated in 197 ICP patients are shown in Table NR1H4 gene in 197 samples with ICP disease, we designed a total of nine pairs of primers . A total of 25 µL PCR system, including 2 µL total 100 ng DNA, 0.5 µL of each forward and reverse primer (2 μM), 12.5 µL mixed comprising Mg2+, dNTPs and Taq polymerase , and 9.5 µL ddH2O were mixed in a reaction tube. Touch down procedures were used for PCR amplification as follows: first, DNA was initially denatured at 94 °C for 5 min, followed by 26 cycles of 94 °C for 30 s, (68–0.5) °C for 30 s, and 72 °C for 45 s, after which 19 thermal cycles for 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, a final extension stage of 72 °C for 10 min, and storage at 4 °C. The obtained PCR products were then examined by 1% agarose gel electrophoresis and sequenced by an ABI 3730 Genetic Analyser . The potential mutations of the NR1H4 gene were detected by comparative analysis of 197 samples with ICP disease and 1029 controls without ICP. The mutation site was searched by bidirectional sequencing.To excavate the potential mutations of the rs Table  to sequeThe evolutionary conservative analysis of p.S145Fand p.M185L were performed in 26 representative species, including Chimpanzee, Gibbon, Macaque, Olive baboon, Gelada, Marmoset, Prairie vole, Mouse, Rat, Alpine marmot, Rabbit, Domestic yak, Cow, Goat, Sheep, Sperm whale, Arabian camel, Chacoan peccary, Pig, Dog, Dingo, Cat, Leopard, Horse and Elephant, through the genomic alignments of the Ensembl Genome Browser.NR1H4 gene were conducted using the SWISS-MODEL repository database (http://www.expasy.org/). Then, we compared the protein models simultaneously with the Chimera 1.14rc package.The protein template of modelling between the reference and modified (p.S145F and p.M185L) mutations of the summary function was used to perform the descriptive statistics on the clinical data of 197 samples with ICP disease. The t.test function was conducted to analyse the potential association of 29 clinical data between ICP samples with or without NR1H4 mutations. The P values were two sided, and the results were considered significantly different at P < 0.05. The frequency significant difference for NR1H4 mutations between 197 ICP samples and databases were analysed by Fisher’s test function. All the analyses were completed with R software. Logistic regression analysis was performed to assess the clinical parameters  with the mutations.The NR1H4 gene and detected a total of four mutations, including three missense mutations in exons 2, 3 and 4 and one synonymous mutation in exon 5 with 3 samples in 197 ICP patients.We sequenced 9 exon fragments of the http://www.internationalgenome.org/), and ExAC (http://exac.broadinstitute.org/) databases. There was a significant difference (P = 0.018) in the frequency for two novel mutations between 197 ICP samples and the ChinaMAP (http://www.mbiobank.com/) database.Two out of three missense mutations were novel novel-1, novel-2)  and the synonymous mutation rs147030757 (P = 1.63e−05) between 197 ICP patients and the ExAC database. In addition, rs147030757 showed a significant frequency difference between the ICP population and 1000G_ALL (P = 0.001).The other missense mutation rs180957965 (p.Ala230Ser) was identified in a 30-year-old sample (ICP12), and the synonymous mutation rs147030757 (p.Asn275Asn) were identified in three ICP patients . These mutations were all absent in the controls and had a low frequency of databases, ranging from 0.00018 to 0.0057. There was a significant difference in the frequency of the missense mutation rs180957965 .The clinical and biochemical features of the six ICPs with 4 mutations are presented in Table Evolutionary conservation analysis showed that these two novel mutations (p.S145F and p.M185L) were highly conserved among the 26 species, ranging from human to elephant  from the Du Q et al. report [NR1H4 was upregulated in the ICP group .To further explore the genetic basis of . report . The resoup Fig. D, even tNR1H4 four mutations and 29 available clinical and laboratory data are presented in Table P = 0.014) higher in the mutation group (139.50 mmol/L) than in the wild-type group (137.27 mmol/L). The associations between the clinical parameters (age: odds ratio (OR) = 0.965; 95% confidence intervals (CI): 0.823–1.132; gestational age ; BMI ; gravidity ; parity  and the mutations were shown by logistic regression analysis.The potential correlation of NR1H4 is required for the basal maintenance of enterohepatic circulation and is responsible for bile acid homeostasis. Milona et al. reported that increased hepatic bile acid concentrations during pregnancy in mice are associated with reduced NR1H4 function [NR1H4 function during pregnancy may be associated with elevated levels of serum bile acids [NR1H4 are associated with ICP disease/progressive familial intrahepatic cholestasis [NR1H4 and conducted cell function analysis [ABCB11 and IBABP. In the present study, the two novel mutations S145F and M185L were also located in the first and second zinc finger of the DBD of NR1H4. The mutant has a slight change in the chemical bond of the structure for the NR1H4 gene compared to the wild-type  and one synonymous mutation (MAF = 0.007) were lower in this study. According to previous studies, low-frequency and rare variants with large effect sizes contribute to complex traits and diseases [NR1H4 mutations even though the mutation group tended to be associated with higher TBA levels when considering the allele frequency and size effect. Except for the ICP caused by the NR1H4 mutations, we speculated that other gene mutations  [To date, an increasing number of researchers have found rare (MAF < 0.01) and low-frequency (0.05 ≤ MAF ≤ 0.01) variants associated with human pregnancy diseases, such as spontaneous preterm birth, cardiomyopathy and preeclampsia, by whole-exome sequencing , 39. Condiseases –42. Therceptors) , 35, 43,ceptors) –46, oestceptors) , hypoxiaceptors)  and the ceptors) , among oceptors) , may be NR1H4 variants had symptomatic gallstones [NR1H4 mutations, we found that the TBA levels, CHOL levels and TG levels were higher than the reference values. Therefore, we speculated that these ICP cases with NR1H4 variants have a high risk for gallstones. Bergheim et al. demonstrated that the possible mechanism of gallstones is the decrease in the expression of the NR1H4 gene [NR1H4 could be associated with cholesterol [NR1H4 dysfunctions may occur during the progression associated with inflammatory bowel disease, colorectal cancer in the gut [Considering that BAs are toxic to the body, the excessive increase in BA levels has been depicted in different pathological contents. Moreover, several previous studies demonstrated that BAs have the ability to promote lipid absorption and biliary cholesterol secretion , 51, 52,llstones , and the1H4 gene . Furtherlesterol . In addi the gut , 56, fib the gut , 58. TheNH1R4 gene, such as M173T, R176* and Tyr139_Asn140insLys, have been identified thus far [NR1H4 mutations have been detected in ICP families [NR1H4 genes of hepatic disease among pregnant patients from a relatively large nationally representative sample (n = 197) in China and 1029 local healthy pregnant women. Third, the 29 clinical data of 197 ICP patients are relatively complete, which provides data supporting correlation analysis between mutations and clinical data. However, even though our results provided possible pathogenic variants, the causality between the two potential interesting candidate loci and ICP disease needs to be verified by validation functional experiments.Our present study had several advantages. First, to our knowledge, only a few pathogenic mutations of the thus far , 21. Ourfamilies , 21. To NR1H4 gene in two out of 197 Chinese patients with ICP for the first time. Our findings provide new insights into the genetic architecture of ICP disease and suggest potential candidate variant targets for ICP clinical treatment.In summary, we reported two potential damaging mutations (p.S145F and p.M185L) in the Additional file 1. DNA sanger sequencing electropherograms of two novel mutations (p.S145F and p.M185L) in the NR1H4 gene.