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ObjectiveTo observe the effect of Banxia Xiexintang （BXT） on the proliferation of human gastric cancer HGC-27， MKN-45， and AGS cells and its mechanism. MethodCell counting kit-8 （CCK-8） was used to detect the effects of different concentrations of BXT-containing serum （5%， 10%， and 20%） on the proliferation of HGC-27， MKN-45， and AGS cells. A mitochondrial membrane potential probe （TMRE） was used to detect the expression of mitochondrial membrane potential in cells. A kit was used to detect iron ion （Fe2+） content， lipid peroxide （LPO）， and superoxide dismutase （SOD） activity. Western blot was used to detect the protein expression levels of glycogen synthase3β （GSK3β）， phosphorylated GSK3β （p-GSK3β）， nuclear factor E2 related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）. The real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of member 11 of the cystine/glutamic acid reverse transporter solute vector family 7 （SLC7A11）， member 2 of the heavy chain solute vector family 3 （SLC3A2）， transferrin receptor 3 （TFRC）， and tumor protein （TP）53. ResultCCK-8 results showed that BXT and capecitabine could significantly reduce the survival rate of three kinds of gastric cancer cells after treatment with drug-containing serum for 24 h （P<0.01）. After 48 h of intervention with drug-containing serum， the survival rate of three kinds of gastric cancer cells was significantly decreased in both the capecitabine group and the BXT group compared with the blank group. The BXT group was dose-dependent， with 20% BXT having the most significant effect （P<0.01）. In terms of biochemical indicators of ferroptosis， compared with the blank group， BXT and capecitabine significantly decreased the expression of mitochondrial membrane potential （P<0.01） and SOD activity （P<0.01） and significantly increased the contents of LPO and Fe2+ （P<0.01）， so as to improve the sensitivity of gastric cancer cells to ferroptosis. In terms of the Nrf2/GPX4 pathway， compared with the blank group， the BXT group could reduce the protein expressions of p-GSK3β， Nrf2， and GPX4 （P<0.01） in gastric cancer cells and increase mRNA expressions of SLC7A11 and SLC3A2 （P<0.05）. It could also increase the protein expression of GSK3β （P<0.01） and mRNA expression of TP53 and TFRC （P<0.05， P<0.01） in gastric cancer cells. Inhibition of the Nrf2/GPX4 pathway induces ferroptosis in gastric cancer cells. Compared with the capecitabine group， the 20% BXT group showed a more obvious effect. ConclusionBanxia Xiexintang can induce ferroptosis in gastric cancer cells HGC-27， MKN-45， and AGS by inhibiting the Nrf2/GPX4 pathway.
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AIM:To investigate the mechanism through which esculetin induces ferroptosis of mouse breast cancer 4T1 cells.METHODS:Molecular docking of esculetin with p53,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)proteins was performed,and Kaplan-Meier curves and time-dependent receiver oper-ating characteristic curves were drawn.The 4T1 cells were divided into 6 groups,namely the control(CON),1/2 IC50,IC50,2 IC50,erastin,and capecitabine groups.The appropriate drug concentration for treating 4T1 cells was screened by CCK-8 assay.The invasion and migration abilities of 4T1 cells following esculetin treatment at the selected drug concentra-tion were analyzed by scratch test and Transwell assay.Mitochondrial morphological change were examined by electron mi-croscopy.The levels of ferroptotic cell death were then quantified by Fe2+,reactive oxygen species(ROS),malondialde-hyde(MDA),glutathione(GSH),and GPX4 assays.Western blot was performed to evaluate the protein levels of p53,SLC7A11,GPX4 and acyl-CoA synthetase long-chain family member 4(ACSL4).RESULTS:Compared with CON group,the esculetin 1/2 IC50,IC50 and 2 IC50 groups showed smaller size,increased membrane density,and decreased ridge of mitochodria,as well as decreased levels of GSH and GPX4,reduced cell invasion and migration abilities,and de-creased levels of SLC7A11 and GPX4 proteins(P＜0.05).Furthermore,these groups exhibited increased levels of Fe2+,ROS,and MDA,as well as elevated p53 and ACSL4 protein abundance(P＜0.05).CONCLUSION:Esculetin pro-motes ferroptosis of 4T1 cells through a mechanism involving the p53/SLC7A11/GPX4 regulatory axis.
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AIM To investigate the protective effects and the mechanism of corosolic acid on doxorubicin-induced cardiotoxicity in H9c2 cardiomyocytes.METHODS To screen and determine the effective concentration of corosolic acid,the injury models of H9c2 cardiomyocytes established by 1 μmol/L doxorubicin were exposed to 24 h different concentrations of corosolic acid,followed by detections of their cell activity by MTT method;their cell apoptosis morphology by Hoechst 33342 staining method;their cell apoptosis rate by Annexin V-FITC/PI double staining method;their intracellular ROS level by DCFH-DA probe;their intracellular iron level by iron ion colorimetry;and their protein expressions of Bax,Bcl-2,cleaved-caspase3,Nrf2,GPX4 and Ptgs2 by Western blot.RESULTS Upon the doxorubicin-induced injury models of H9c2 cardiomyocytes,corosolic acid improved their viability and survival rate(P<0.05),decreased their levels of ROS and Fe2+ and the apoptosis rate(P<0.05),up-regulated the protein expressions of Bcl-2,Nrf2 and GPX4(P<0.05),and down-regulated the protein expressions of Bax,cleved-caspase 3 and Ptgs2(P<0.05).CONCLUSION Corosolic acid can inhibit the ROS level and apoptosis of doxorubicin-induced injury models of H9c2 cardiomyocytes,and the iron death as well via activating Nrf2/GPX4 pathway.
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Objective @#To investigate the expression of genes and proteins in the peripheral blood mononuclear cell ( PBMC) cystine / glutamate antiporter system (System Xc-) / glutathione ( GSH) / glutathione peroxidase 4 ( GPX4) ferroptosis pathway and its influence on inflammatory factors in patients with rheumatoid arthritis ( RA) ．@*Methods @#30 patients with RA and 30 healthy participants were enrolled．PBMCs were isolated using Ficoll-hypaque density gradient centrifugation．The cells were categorized into the healthy control，RA，ferroptosis inhibitor，ferroptosis in- ducer group．The cell viability was checked using the cell counting kit-8 ( CCK-8) method．Intracellular Fe2 + rela- tive fluorescence intensity and reactive oxygen species (ROS) levels were detected using the FerroOrange and Di- hydroethidium (DHE) fluorescent probes，respectively．Western blot and real-time quantitative PCR (qPCR) de- tected the expression of nuclear factor erythroid 2-related factor 2 ( Nrf2 ) ，solute carrier family 7 member 11 (SLC7A11) ，GPX4 proteins and mRNA．And the flow cytometry quantified the levels of tumor necrosis factor α (TNF-α) ，Interleukin (IL) -1，and IL-6 in the supernatant of each cell group. @*Results @# Compared to the healthy control group，the RA group showed a significantly increased Fe2 + concentration and elevated ROS levels，reduced expression of Nrf2，SLC7A11 and GPX4 proteins and mRNA，and increased contents of TNF-α , IL-1 and IL-6 in PBMC supernatant，and the differences were statistically significant．The concentration of Fe2 + and ROS levels in the inhibitor group were lower than those in the RA group，the proteins expressions of Nrf2，SLC7A11 and GPX4 increased，the mRNA expressions of SLC7A11 and GPX4 increased，the content of IL-6 in the PBMC supernatant decreased but the content of TNF-α increased，and the differences were statistically significant．In contrast，the in- ducer group，when compared to the RA group，displayed increased ROS levels，reduced expression of SLC7A11 protein and mRNA and decreased expression of Nrf2 protein，and the contents of TNF-α and IL-1 in the PBMC su- pernatant increased，but the expression of GPX4 protein increased ，and the differences were statistically signifi- cant．The inducer group，compared to the RA group，showed increased cell viability，and the difference was statis- tically significant (P＜0. 000 1) ．@*Conclusion @# The presence of ferroptosis in PBMC in RA patients，inhibiting or inducing PBMC ferroptosis in RA patients，will inhibit or promote the secretion of inflammatory factors．Inhibition of PBMC ferroptosis in RA patients may be helpful in the treatment of RA.
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Objective To investigate the effects of indirubatin derivative E804 on proliferation and migration of non-small cell lung cancer(NSCLC)A549 cells,and to elucidate the possible mechanism of Nrf2-HO-1/GPX4 pathway.Methods Lung cancer A549 cells were used as the cell model.The proliferation and migration of differ-ent specific inhibitors(Nec-1,CQ,Z-VAD,DFO,Fer-1 and Lip-1)in 0,10 μmol/L E804 and 10 μmol/L E804+groups were observed by MTT and cell scratch assay.The contents of reactive oxygen species(ROS)were de-tected by DCFH-DA fluorescence probe method,the contents of Fe2+were detected by colorimetric method,the contents of reduced glutathione(GSH)were detected by spectrophotometry,and the contents of malondialdehyde(MDA)were detected by micromethod.The expression levels of SLC7A11,Transferrin,GPX4,SLC40A1,Nrf2 and HO-1 were detected by Western blot in cells of 0,2.5,5 and 10 μmol/L E804 groups.Results Compared with the control group(0 μmol/L E804),2.5,5 and 10 μmol/L E804 significantly increased intracellular ROS,Fe2+and MDA levels,and decreased intracellular GSH content(P＜0.01).Meanwhile,the expression levels of SLC7A11,GPX4,SLC40A1,Nrf2 and HO-1 significantly decreased(P＜0.01),and the expression level of Transferrin increased(P＜0.05).Compared with the 10 μmol/L E804 group alone,the apoptosis inhibitor(Z-VAD)group and the ferroptosis inhibitor(DFO,Fer-1 and Lip-1)group could significantly reverse the inhibition of proliferation and migration of A549 cells by 10 μmol/L E804(P＜0.01).Conclution E804 can induce ferrop-tosis and inhibit the proliferation and migration of A549 cells,which may be related to the inhibition of Nrf2-HO-1/GPX4 pathway.
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Purpose To investigate the effect of autophagy intervention on ferroptosis and drug resistance of colorectal canc-er cells and its molecular mechanism.Methods The human colorectal cancer cell lines HCT-8,COLO205,HCT-116,SW620,and SW480 were cultured.HCT-116 cells with moder-ate expression of LC3 were screened,and the expression differ-ences of LC3,p62,Keap1,Nrf2,GPX4 proteins,Fe2+,GSH,and MDA between them and OXA-resistant HCT-116/OXA cell lines were detected.The expression levels of LC3,p62,Keap1,Nrf2,GPX4,Fe2+,GSH and MDA were assessed in HCT-116/OXA cells through the intervention of autophagy and ferroptosis intervention agent combined with oxaliplatin.The proliferative activity and sensitivity to oxaliplatin in each group were detected by CCK-8 assay.Cell growth and invasion ability of each group were detected by plate cloning and Trans well assay.Results LC3,p62 and GPX4 expression levels of HCT-116 cells in the 5 groups were moderate.Compared with HCT-116 cells,HCT-116/OXA was less sensitive to oxaliplatin,and the proteins of p62,Nrf2 and GPX4 were highly expressed,LC3 and Keap1 were lowly expressed,and the expression of Fe2+,GSH and MDA were increased(P＜0.05).The levels of LC3,Keap1 protein,Fe2+and MDA in Rapa and Rapa+Fer-1 groups were higher than those in Fer-1 and control groups,while p62,Nrf2,GPX4 and GSH levels were lower.The expressions of GPX4 pro-tein and GSH in Rapa+Fer-1 group were lower than those in Rapa group(P＜0.05).In the autophagy inhibitor group,LC3,p62,Nrf2,GPX4 and GSH were highly expressed in the CQ and CQ+Erastin groups compared with the control and Eras-tin groups,while Keap1 protein,Fe2+and MDA were low.The levels of GPX4 protein and GSH in Erastin group were lower than those in the other three groups,and the levels of Fe2+and MDA were higher than those in the other three groups(P＜0.05).The combination of autophagy activator OXA showed that Rapa intervention group had higher chemical sensitivity to OXA,less number of migrating cells and lower cell proliferation activity than the other three groups.The sensitivity of Rapa+Fer-1 group to oxaliplatin was lower than that of Rapa group,but higher than that of Fer-1 group and control group(P＜0.05).There was no significant difference between Fer-1 group and con-trol group(P＜0.05).Compared with the control group,the cell activity,migration capacity and clonogenesis capacity of Erastin,CQ+Erastin and CQ groups were decreased when auto-phagy inhibitor was combined with OXA,and the Erastin group was the lowest,while the CQ+Erastin group was higher than the Erastin group,and lower than the CQ group(P＜0.05).Con-clusion In colorectal cancer,autophagy is involved in the regu-lation of ferroptosis,and intervention in autophagy can regulate ferroptosis in colorectal cancer cells through the p62-Keap1/Nrf2-GPX4 pathway,thereby reversing oxaliplatin resistance.
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ObjectiveTo investigate the effect of Zuoguiwan on the ovarian function in the rat model of cyclophosphamide-induced premature ovarian failure （POF） based on the changes of ferroptosis pathway. MethodForty SD rats were randomized into blank， model， and low- and high-dose （2， 8 g·kg-1， respectively） Zuoguiwan groups， with 10 rats in each group. The rats in the other groups except the normal group were intraperitoneally injected with CTX at a dose of 50 mg·kg-1 on the first day and 8 mg·kg-1 from the second day to the fifteenth day for the modeling of POF. After modeling， the rats were administrated with corresponding drugs or normal saline by gavage for four weeks. Hematoxylin-eosin staining was performed to observe the pathological changes in the ovarian tissue. The mitochondria of the ovarian tissue was observed by electron microscopy. The serum levels of follicle-stimulating hormone （FSH）， estradiol （E2）， luteinizing hormone （LH）， anti-Mullerian hormone （AMH）， malondialdehyde （MDA）， catalase （CAT）， superoxide dismutase （SOD）， and iron ion were measured by biochemical methods and enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of glutathione peroxidase 4 （GPX4）， solute carrier family 7 member 11 （SLC7A11）， ferritin heavy chain （FTH1）， and acyl-CoA synthetase long chain family member 4 （ACSL4）. ResultCompared with the blank group， the model group showcased significantly increased atretic follicles， atrophied， fragmented， and vacuolated mitochondria， and reduced， loose， and disordered cristae in mitochondria. Compared with the model group， high-dose Zuoguiwan increased mature follicles， the volume of mitochondria in the ovary， alleviated the vacuolation， and improved the number and arrangement of mitochondrial cristae. Compared with the blank group， the modeling elevated the levels of iron， MDA， FSH， and LH， up-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， decreased the activities of SOD and CAT， lowered the levels of E2 and AMH， and down-regulated the expression of ACSL4 （P<0.05， P<0.01）. Compared with the model group， drug interventions lowered the levels of iron， MDA， FSH， and LH， down-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， increased the activity of CAT， elevated the levels of E2 and AMH， and up-regulated the expression of ACSL4 （P<0.05， P<0.01）. ConclusionZuoguiwan may inhibit the occurrence of ferroptosis by regulating the SLC7A11/GPX4 axis， thereby improving the ovarian function of POF rats.
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ObjectiveTo investigate the protective effect of Zhuangtongyin on the Middle Cerebral Artery Occlusion （MCAO） model by modulating ferroptosis through the Nrf2-SCL7A11/xCT-Gpx4 pathway and its underlying mechanism. MethodsC57BL/6J mice were randomly divided into Sham operation group （Sham）， model group （MCAO）， low-dose Zhuangtongyin group （ZTY-L）， high-dose Zhuangtongyin group（ZTY-H）， with 5 mice in each group. The MCAO group was modelled by silica gel embolization， the middle cerebral artery of mice was embolized for 1h， then the silica gel was pulled out and reperfusion was performed after 72 h； and the other operations in the Sham group were the same as those in the MCAO group except that the thread plug was not inserted. The neural function of mice was evaluated by Zea-Longa method. TTC staining was used to evaluate the volume of cerebral infarction. The level of brain injury was evaluated by HE staining and Nissl staining. Prussian blue staining and the expression of iron transport-related carrier receptors TfR1 and DMT1 on mRNA level was detected by qPCR to evaluate the iron ion deposition level in mice brain. The expression of lipid peroxidation-related gene ACSL4 on mRNA level was detected by qPCR， and the content of 4-HNE was detected by ELISA kit to evaluate the lipid peroxidation level of mice brain. The expressions of ferroptosis marker PTGS2 mRNA level was detected by qPCR. The expressions of Nrf2， SCL7A11/xCT， Gpx4 in mice brain tissue were detected by Western-blot and immunofluorescence. ResultsZhuangtongyin improved the nerve function of mice after MCAO （P＜0.05） and the cerebral infarction volume of mice （P＜0.05） and alleviate the pathological injury of cerebral cortex cells after MCAO operation. Zhuangtongyin attenuated the accumulation of trivalent iron ions in the brain tissue of mice following MCAO. Additionally， Zhuangtongyin downregulated the expression of TfR1 and DMT1 mRNA （P＜0.001）， a transporter associated with cellular iron ion uptake， in the brains of post-MCAO mice. Furthermore， Zhuangtongyin reduced levels of lipid peroxidation product 4-HNE （P＜0.001） and suppressed ACSL4 mRNA expression in brain tissue post-MCAO （P＜0.001）. Besides， Zhuangtongyin downregulated the expression of PTGS2 mRNA （P＜0.001）， in the brains of post-MCAO mice. Zhuangtongyin increased the expression of nrf2 into the nucleus （P＜0.001）， and increased the expression of xCT and Gpx4 in neurons after MCAO （P＜0.001）. ConclusionZhuangtongyin can enhance the nerve function and reduce cerebral infarction volume in MCAO/R mice， alleviate the pathological damage of cerebral cortex cells， and modulate the expression of key signaling molecules in the Nrf2-SCL7A11/xCT-Gpx4 pathway. Therefore， it is suggested that the mechanism by which Zhuangtongyin improves MCAO/R injury in mice may involve regulating ferroptosis through the Nrf2-SCL7A11/xCT-GPX4 pathway.
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BACKGROUND:Synovitis plays a leading role in the early progression of knee osteoarthritis and is a potential early therapeutic target.However,the mechanism of synovitis remains unclear.In animal models,increased systemic iron and intracellular iron uptake can induce and exacerbate osteoarthritis,but the association with ferroptosis in synovitis remains unclear.Further studies are needed to investigate the role of ferroptosis in the development and progression of synovitis in osteoarthritis. OBJECTIVE:To investigate the role of ferroptosis mediated by glutathione peroxidase 4(GPX4)in the development of synovitis in knee osteoarthritis. METHODS:The synovial tissues of 43 patients with osteoarthritis who underwent arthroscopic or joint replacement surgery and 10 patients undergoing arthroscopic treatment of meniscal injury or ligament tear were collected and divided into three groups according to the Kellgren-Lawrence grading of X-ray images:normal control group(KLG 0,n=10),early knee osteoarthritis group(KLG 1,2,n=20)and late knee osteoarthritis group(KLG 3,4,n=23).Hematoxylin-eosin staining was used to observe the severity of synovitis in each group,and iron deposition in the synovium in each group was evaluated by Prussian blue staining.The expressions of Acyl-CoA Synthetase Long Chain Family Member 4(ACSL4),GPX4,cyclooxygenase 2 and tumor necrosis factor α in the synovium were detected by immunohistochemical staining.Western blot and immunofluorescence were used to detect the expression of ferroptosis-related proteins ACSL4 and GPX4. RESULTS AND CONCLUSION:Compared with the normal control group,iron content in synovial tissue was increased in the knee osteoarthritis groups,and iron deposition in the late knee osteoarthritis group was higher than that in the early osteoarthritis group.ACSL4 was highly expressed in the synovial tissue of knee osteoarthritis compared with the normal control group(P＜0.01),and GPX4 was lowly expressed in the synovial tissue of knee osteoarthritis(P＜0.01).The expression level of ACSL4 increased with the progression of osteoarthritis,while the expression level of GPX4 decreased with the progression of osteoarthritis.The expression of cyclooxygenase 2 in the synovium of osteoarthritis was significantly higher than that in the normal synovium,and the expression was the highest in the early stage of osteoarthritis,which was significantly different from that in the advanced stage of osteoarthritis(P＜0.01).The expression of tumor necrosis factor α in the synovium of osteoarthritis was significantly higher than that in the normal synovium,but there was no significant difference between early and late osteoarthritis groups(P＞0.05).To conclude,the deposition of iron exists in the synovial tissue of osteoarthritis and ferroptosis is involved in the occurrence and progression of synovitis in knee osteoarthritis.
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BACKGROUND:Studies have shown that imbalance of bone metabolism during glucocorticoid-induced osteonecrosis of the femoral head necrosis is closely related to oxidative stress. OBJECTIVE:To investigate the pathological mechanism by which oxidative stress-induced ferroptosis promote apoptosis in osteoblasts involved in steroid-induced osteonecrosis of the femoral head. METHODS:General data and serum specimens were collected from 47 patients with steroid-induced osteonecrosis of the femoral head.In addition,six femoral head specimens were collected from these patients.According to the Association Research Circulation Osseous(ARCO)staging system,serum specimens were grouped into ARCO Ⅱ,Ⅲ,and IV,while femoral head specimens were classified into ARCO Ⅲ and IV.Serum levels of malondialdehyde and superoxide dismutase 1 were measured.The protein expression of superoxide dismutase 1,glutathione peroxidase 4,Bcl-2 in the femoral head was detected and verified by Data independent acquisition(DIA)for quantitative sequencing,western blot and alkaline phosphate detection. RESULTS AND CONCLUSION:The ARCO stage of patients with steroid-induced osteonecrosis of the femoral head was independent of age,sex and necrotic side.The serum levels of malondialdehyde and superoxide dismutase 1 were higher in patients with ARCO stage Ⅲ compared with those with ARCO stage Ⅱ and IV.The results of DIA protein quantification showed that the function of differential proteins was mainly related to redox.The levels of superoxide dismutase 1,glutathione peroxidase 4,and Bcl-2 in the necrotic region were lower than in the normal region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.Western blot verified the results of DIA protein quantification.The alkaline phosphatase activity was lower in the necrotic region than in the normal region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.In the necrotic and sclerotic regions,the function of differential proteins was also related to redox,and superoxide dismutase 1,glutathione peroxidase 4,Bcl-2 protein expression and alkaline phosphatase activity were lower in the necrotic area than in the sclerotic region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.To conclude,glucocorticoids can influence the progression of steroid-induced osteonecrosis of the femoral head by upregulating oxidative stress levels,inducing osteoblast ferroptosis,and inhibiting osteogenic function.
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BACKGROUND:Disturbances in bone metabolism have a significant association with ferroptosis in steroid-induced osteonecrosis of the femoral head(SONFH).Furthermore,the pathologic process of SONFH is characterized by the presence of cartilage damage and degeneration.However,the specific regulatory targets and the relationship between ferroptosis and cartilage concerning SONFH remain unclear. OBJECTIVE:To employ bioinformatics and machine learning techniques to identify specific genes associated with ferroptosis that target cartilage and to investigate the correlation between ferroptosis and cartilage,thereby providing novel ideas and methodologies for the study and treatment of SONFH. METHODS:Disease datasets pertinent to the study and ferroptosis-related genes were retrieved from the GEO and FerrDb databases.Subsequently,the disease datasets were normalized and differential analysis using the R language to identify ferroptosis-related differential genes(Fe-DEGs).We conducted Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis of Fe-DEGs.Furthermore,ferroptosis-related signature genes were filtered based on the protein-protein interaction network of Fe-DEGs and machine learning methods.Finally,the rabbits were divided into normal and model groups.The normal group was given the same dose of saline to simulate the modeling drug,and the animal model of SONFH in rabbits was constructed by injection of modified horse serum combined with methylprednisolone.After successful modeling,the expression of signature gene was verified between different groups,and the phenotype of ferroptosis in cartilage was analyzed. RESULTS AND CONCLUSION:Through the normalization and differential analysis of the dataset,a total of 1 315 differentially expressed genes were identified.Additionally,379 ferroptosis-related genes were obtained from the FerrDb database.After intersecting both gene sets,19 Fe-DEGs were obtained.The GO analysis revealed that Fe-DEGs were mainly involved in biological processes such as cell migration and cellular response to oxidative stress,cellular components such as kinase complexes,amino acid complexes,and cytoplasmic membranes,as well as molecular functions such as kinase activity,receptor activity,and protein binding.The KEGG analysis revealed that Fe-DEGs were mainly enriched in the FoxO signaling pathway,vascular endothelial growth factor signaling pathway,and FcγR-mediated phagocytosis.Constructing a protein-protein interaction network and using machine learning,we identified the ferroptosis-related signature gene,CA9.The gene set enrichment analysis of the signature gene CA9 revealed an upregulated expression in biological processes such as fatty acid metabolism and O-GlcNAc glycosylation modification,while being inhibited in terms of neural activity and ligand-receptor interactions.RT-PCR and western blot results showed that compared with the normal group,the expressions of ACSL4 and CA9 at mRNA and protein levels were significantly higher in the model group(P<0.05),while the expressions of SLC7A11 and GPX4 at mRNA and protein levels were significantly lower in the model group(P<0.05),coinciding with the expression levels of the signature genes in the dataset.These findings indicate that the cartilage of SONFH is closely related to ferroptosis,and targeting the signature gene may provide certain ideas and directions for the study and treatment of SONFH.
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BACKGROUND:Ferroptosis-mediated ischemia-reperfusion injury plays a crucial role in the occurrence and progression in pressure ulcers,and there may be pressure ulcer-associated ferroptosis biomarkers,but the mechanism has not been elucidated. OBJECTIVE:To investigate the molecular mechanisms underlying pressure ulcers using bioinformatic analysis,with a focus on identifying differentially expressed genes associated with ferroptosis during the process of pressure ulcer formation,thereby providing novel insights into the clinical treatment of pressure ulcers. METHODS:The single-cell transcriptome sequencing dataset and ferroptosis-related genes were obtained and preprocessed from the Gene Expression Omnibus(GEO)and FerrDb databases.We performed clustering and proportion analyses,metabolic activity and pseudotime analysis,cell communication analysis,ferroptosis gene set cell population identification,and enrichment analysis to determine differentially expressed genes related to ferroptosis.Animal experiments were then conducted for further validation,with 20 Sprague-Dawley rats randomly assigned into a control group and a model group(n=10 per group).The control group received no treatment,while the model group underwent a cycle of ischemia-reperfusion to establish pressure ulcer models.Changes in differentially expressed genes and proteins in the wound tissues of pressure ulcer rats were detected using fluorescent quantitative PCR and western blot,respectively. RESULTS AND CONCLUSION:The single-cell transcriptome sequencing data were clustered into six cell types,with a higher proportion of type 2 and type 3 keratinocytes observed in the pressure ulcer group.There was evident metabolic heterogeneity and evolutionary trajectory among cell populations.Type 2 and type 3 keratinocytes exhibited stronger cell communication,while type 2 keratinocytes demonstrating optimal ligand-receptor interactions.Type 2 keratinocytes demonstrated higher scores for ferroptosis,accompanied by significant upregulation or downregulation of specific genes.A total of 27 Gene Ontology enrichments,20 Kyoto Encyclopedia of Genes and Genomes enrichments,and 24 ferroptosis-related differentially expressed genes,including glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long chain family member 4(ACSL4),were identified.Animal experiments further confirmed the downregulation of GPX4,the ferroptosis-inhibiting protein,and the upregulation of ACSL4,the ferroptosis-promoting protein,in the model group.Overall,these findings indicate the presence of ferroptosis in pressure ulcer tissue.GPX4 and ACSL4 are important genes regulating ferroptosis in pressure ulcer tissues.
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ObjectiveTo investigate the effect and mechanism of Zhenwutang on renal oxidative damage in the mouse model of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency via the nuclear factor erythroid 2-related factor-2 （Nrf2）/heme oxygenase-1 （HO-1）/glutathione peroxidase 4 （GPX4） signaling pathway. MethodTwenty-five 7-week-old SPF-grade male db/m mice and 95 7-week-old SPF-grade male db/db mice were adaptively fed for a week. A blank group was set with the db/m mice without treatment， and the other mice were administrated with Rhei Radix et Rhizoma decoction and hydrocortisone for the modeling of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency. The modeled mice were randomized into the model， irbesartan （25 mg·kg-1）， and high-， medium-， low-dose （33.8， 16.9， 8.45 g·kg-1） Zhenwutang groups （n=15） and administrated with corresponding drugs for 8 weeks. The survival status of mice was observed， and the traditional Chinese medicine （TCM） syndrome score was recorded. The indicators related to spleen-kidney Yang deficiency， fasting blood glucose （FBG）， and renal function indicators were determined. Hematoxylin-eosin staining was employed to observe the histopathological changes of the renal tissue in each group. Biochemical kits were used to determine the oxidative stress-related indicators in the renal tissue. Real-time polymerase chain reaction and Western blotting were employed to determine the mRNA and protein levels， respectively， of Nrf2， HO-1， glutamate-cysteine ligase catalytic subunit （GCLC）， and GPX4 in the renal tissue of mice in each group. ResultCompared with the blank group， the modeling increased the TCM syndrome score （P<0.05）， elevated the estradiol （E2） and FBG levels （P<0.05）， lowered the testosterone （T）， triiodothyronine （T3）， and tetraiodothyronine （T4） levels （P<0.05）， and weakened the renal function （P<0.05）. In addition， the modeling led to glomerular hypertrophy and glomerular mesangial and basal thickening， decreased the catalase （CAT） activity， total antioxidant capacity （T-AOC）， and glutathione （GSH） content （P<0.05）， increased the malondialdehyde （MDA） content （P<0.05）， and down-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. Compared with the model group， high and medium doses of Zhenwutang decreased the TCM syndrome score and E2 content （P<0.05）， increased the T， T3， and T4 content （P<0.05）， improved the renal function （P<0.05）， alleviated the pathological changes in the renal tissue， increased CAT， T-AOC， and GSH （P<0.05）， reduced MDA （P<0.05）， and up-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. ConclusionZhenwutang can improve the general state and renal function and reduce the oxidative damage and pathological changes in the renal tissue of db/db mice with spleen-kidney Yang deficiency by regulating the Nrf2/HO-1/GPX4 signaling pathway.
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OBJECTVE To study the improvement effects of obacunone on renal interstitial fibrosis （RIF） in unilateral ureteral obstruction （UUO） model mice， and to investigate its mechanism based on ferroptosis mediated by nuclear factor erythroid 2- related factor 2（Nrf2）/glutathione peroxidase 4 （GPx4） signaling pathway. METHODS Thirty mice were randomly divided into sham operation group， model group， irbesartan group （positive control， 20 mg/kg）， obacunone low-dose and high-dose groups （10， 40 mg/kg）， with 6 mice in each group. Except for sham operation group， UUO model was established by ligation of unilateral ureter in other groups. After operation， administration groups were given intraperitoneal injection of relevant medicine， and sham operation group and model group were given intraperitoneal injection of constant volume of normal saline， once a day， for 7 consecutive days. The levels of creatinine （Cr） and blood urea nitrogen （BUN）， the content of malondialdehyde （MDA） and the activities of total superoxide dismutase （T-SOD） in serum and the concentration of Fe2+ in renal tissue were all detected. HE staining and Masson staining were performed to observe the morphology and the fibrosis of renal tissues. Immunohisto- chemical staining was used to determine expressions of the fibronectin （Fn）， α-smooth muscle actin （α-SMA）， GPx4 964083717@qq.com and Nrf2 in renal tissue. Western blot and real-time quantitative polymerase chain reaction （RT-qPCR） were used E-mail：834300205@qq.com to detect the protein and mRNA levels of Fn， α-SMA， Nrf2， GPx4 and SLC7A11 in the renal tissues. RESULTS Compared with sham operation group， serum levels of Cr and BUN， the concentration of Fe2+ in renal tissue， the protein and mRNA levels of Fn and α-SMA in model group were increased significantly （P＜0.05）， while the activity of T-SOD in serum， protein and mRNA expressions of Nrf2， GPx4， SLC7A11 in kidney tissue were significantly decreased （P＜0.05）； in the kidney tissue， the renal tubules were dilated， the collagen deposition was obvious， the fibrous bands were thicker and darker， and the renal interstitial inflammatory cells infiltrated significantly. After intervened with obacunone， the levels of above indexes （except for mRNA expression of SLC7A11 in obacunone low-dose group） in serum and renal tissue were reversed significantly （P＜0.05）， and pathological damage and collagen deposition of kidney tissue were alleviated. CONCLUSIONS Obacunone can improve renal interstitial fibrosis of UUO model mice， the mechanism of which may be associated with activating the Nrf2/GPx4 pathway and then inhibiting ferroptosis to relieve RIF in UUO model mice.
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Ex vivo culture-amplified mesenchymal stem cells (MSCs) have been studied because of their capacity for healing tissue injury. MSC transplantation is a valid approach for promoting the repair of damaged tissues and replacement of lost cells or to safeguard surviving cells, but currently the efficiency of MSC transplantation is constrained by the extensive loss of MSCs during the short post-transplantation period. Hence, strategies to increase the efficacy of MSC treatment are urgently needed. Iron overload, reactive oxygen species deposition, and decreased antioxidant capacity suppress the proliferation and regeneration of MSCs, thereby hastening cell death. Notably, oxidative stress (OS) and deficient antioxidant defense induced by iron overload can result in ferroptosis. Ferroptosis may inhibit cell survival after MSC transplantation, thereby reducing clinical efficacy. In this review, we explore the role of ferroptosis in MSC performance. Given that little research has focused on ferroptosis in transplanted MSCs, further study is urgently needed to enhance the in vivo implantation, function, and duration of MSCs.
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Humans , Antioxidants/metabolism , Ferroptosis , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Iron Overload/metabolismABSTRACT
OBJECTIVE To investigate whether matrine exerts improvement effect on experimental autoimmune encephalomyelitis （EAE） mice by regulating ferroptosis pathway. METHODS Totally 30 female C57BL/6 mice were randomly assigned into normal group， model group and matrine group， with 10 mice in each group. Model group and matrine group were given antigen emulsion containing inactivated Mycobacterium tuberculosis and MOG35-55 to induce EAE model. Matrine group was injected with Matrine injection （50 mg/kg） intraperitoneally since the 7th day after immunization； normal group and model group were given constant volume of normal saline intraperitoneally， once a day， since 18th day after immunization. The neurofunctional score of mice was recorded， and hematoxylin and eosin staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination in spinal cord tissue. The quantitative reverse transcription PCR and Western blot assay were performed to determine the mRNA expressions of transferrin receptor 1 （TFR1）， nuclear receptor coactivator 4 （NCOA4） and hephaestin （Heph）， and the protein expressions of system Xc－ （xCT） and glutathione peroxidase 4 （GPx4）. RESULTS Compared with normal group， accumulative neurofunctional score was significantly increased in model group （P＜0.01）； inflammatory cell infiltration and demyelination were obvious in spinal cord tissue， and related scores were increased significantly （P＜0.01）. The mRNA expressions of TFR1 and NCOA4 in myelin tissue were up-regulated significantly， while the mRNA expression of Heph and the protein expressions of xCT and GPx4 were down-regulated significantly （P＜0.05 or P＜0.01）. Compared with model group， above indexes of matrine group were all improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Matrine can improve EAE mice， the mechanism of which may be associated with regulating iron metabolism pathway and xCT/GPx4 pathway in ferroptosis.
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This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.
Subject(s)
Rats , Male , Animals , Rats, Sprague-Dawley , NF-E2-Related Factor 2/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Triterpenes/pharmacology , Oxidative Stress , Arthritis, Rheumatoid/genetics , Glutathione , Superoxide Dismutase/metabolism , Glycosides/pharmacology , RNA, Messenger/metabolismABSTRACT
This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.
Subject(s)
Humans , Ferroptosis , NF-E2-Related Factor 2/genetics , Reactive Oxygen Species , Epithelial Cells , Antioxidants , Glutathione , GlucoseABSTRACT
Objective:To observe the effect of α-lipoic acid(ALA) on the intracerebroventricular injection(icv) of streptozotocin(STZ)-induced spatial learning memory impairments in rats and the underlying molecular mechanisms.Methods:Forty-five male SD rats were assigned into 3 groups, control group, icv-STZ group and icv-STZ+ ALA group, 15 rats each. STZ was dissolved in artificial cerebrospinal fluid then injected into the lateral ventricles of the rat by using stereotaxic device. ALA was administrated by gavage after STZ injection. The spatial learning memory was examined by using Morris water maze test after 4 weeks of treatment. Immunohistochemistry was performed to detect the number of microglia and astrocytes, electron microscopy was applied to detect mitochondrial integrity, Western blotting was used to detect the protein expression levels, and the changes of lipid peroxidation and redox system were examined by kit.Results:Spatial learning memory was impaired in rats after 4 weeks of STZ injection, and ALA treatment ameliorated STZ-induced cognitive dysfunction in rats. Iron concentration, lipid peroxidation, neuroinflammation, Tau hyperphosphorylated were enhanced markedly after STZ injection, along with and the activation of MAPK and GSK-3β, which were ameliorated by ALA. Further examination revealed that STZ activated the JAK2/STAT3 pathway and transcriptionally inhibited the expression of peroxidase GPX. Inhibition of STAT3 activity can block STZ-induced downregulation of GPX4 and Tau hyperphosphorylation.Conclusion:ALA ameliorated STZ-induced spatial learning memory impairments in rats via deactivation of JAK2/STAT3 pathway, restored GPX4 protein level, resulting in chelating iron, improving mitochondrial function, balancing the redox system, ameliorating Tau hyperphosphorylation and neuroinflammation.
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Objective:To investigate the effect of luteolin(Lut)on the proliferation and ferroptosis of human adriamycin(ADR)resistant chronic myeloid leukemia(CML)K562/ADR cells and the underlying molecular mechanism. Methods:K562 and K562/ADR cells were treated with different concentrations of ADR.The sensitivity of K562 and K562/ADR cells to ADR was evaluated by CCK-8 assay;the effect of different concentrations of Lut on the proliferation of K562/ADR cells was assessed by CCK-8 assay,and the half inhibitory concentration(IC50)was calculated for subsequent experiments;the morphological changes of the cells were observed by an inverted microscope;CCK-8 assay was used to examine the sensitizing effect of Lut on ADR;FCM assay was used to study the effect of Lut on the apoptosis of K562/ADR cells;fluorescence probe DCFH-DA,Fe2+colorimetric assay and glutathione(GSH)kit were used to detect the content of reactive oxygen species(ROS),Fe2+and GSH in K562/ADR cells,respectively;Western blotting was used to examine the expression levels of glutathione peroxidase 4(GPX4),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)in K562/ADR cells;the effects of Lut on the proliferation of K562/ADR cells,ROS content,GSH content,Fe2+content and GPX4 expression were studied after treatment with ferroptosis inhibitor Ferrostatin-1(Fer-1). Results:Compared with control group(cells treated with 0 μmol/L Lut),Lut significantly inhibited the proliferation of K562/ADR cells(P＜0.001),improved the chemosensitivity of K562/ADR cells to ADR(P＜0.05),increased apoptosis rate(P＜0.001)and ROS level(P＜0.05)of K562/ADR cells,reduced the GSH level(P＜0.001),and increase Fe2+content(P＜0.01).Compared with control group(cells treated with 0 μmol/L Lut),the protein expressions of GPX4,Nrf2 and HO-1 decreased with the increase of Lut concentration(P＜0.05).Compared with the Lut treatment alone,the inhibitory effect on the proliferation of K562/ADR cells induced by Lut was partially restored by Fer-1 intervention(P＜0.05),and intracellular ROS level was decreased(P＜0.001),GSH level was increased(P＜0.001),Fe2+content was decreased(P＜0.001)and the expression of GPX4 was increased(P＜0.01)in K562/ADR cells. Conclusion:Lut can inhibit the proliferation of K562/ADR cells through ferroptosis pathway,improve the chemosensitivity to ADR,and the potential mechanism may be related to the Nrf2/HO-1 signaling pathway,which provides experimental basis for the treatment of leukemia by ferroptosis.