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Objective To investigate the mechanism of iron death induced by TRPC6/NF-κB in glomerular podiocytes mediated by high homocysteine(Hcy).Methods Mouse glomerulopocytes were cultured in vitro and divided into Control group(0 μmol/L Hcy)and Hcy group(80 μmol/L Hcy).After 48h of intervention,Western blot was used to detect the expression levels of iron death related proteins GPX4 and SLC7A11 and TRPC6 and NF-κ B.Real-time quantitative fluorescence PCR(qRT-PCR)and immunofluorescence were used to detect the expression of TRPC6.The level of podocyte apoptosis was detected by flow cytometry.Malondialdehyde(MDA)assay kit was used to determine intracellular MDA levels.After transfection of TRPC6 interference fragment and TRPC6 negative control(NC),qRT-PCR was divided into Control,si-NC and si-TRPC6(Si-TRPC6-1,Si-TRPC6-2,Si-TRPC6-3).Western Blot was divided into Control,Hcy,si-NC+Hcy,si-TRPC6+Hcy.The expression of TRPC6 mRNA was detected by qRT-PCR.The expression levels of GPX4,SLC7A11,NF-κB and TRPC6 were detected by Western Blot.The level of podocyte apoptosis after interference was detected by flow cytometry.Results(1)Compared with Control group,the expression levels of iron death related proteins GPX4 and SLC7A11 in Hcy group were decreased,and the apoptosis rate was increased(P<0.05).(2)Compared with Control group,TRPC6 protein,mRNA levels and immunofluorescence expression were increased in Hcy group.The level of MDA and the expression of NF-κB signaling pathway protein increased in Hcy group,and the comparison between the two groups had statistical significance(P<0.05).(3)Compared with the si-NC group,the mRNA expression level of TRPC6 in si-TRPC6(Si-TRPC6-1,Si-TRPC6-2,Si-TRPC6-3)group was decreased,and the interference effect of Si-TRPC6-3 was the best(P<0.05).After transfecting TRPC6 NC and TRPC6 interference fragment and administering Hcy,there was no difference in GPX4,SLC7A11,NF-κB and TRPC6 expression in si-NC+Hcy group compared with Hcy group.Compared with the si-NC+Hcy group,the si-TRPC6+Hcy group had higher expression of iron death related proteins,GPX4 and SLC7A11,lower expression of NF-κB and TRPC6,and decreased apoptosis rate(P<0.05).Conclusion This study confirmed that TRPC6/NF-κB can regulate iron death of renal podocytes under the induc-tion of Hcy,which is one of the mechanisms leading to kidney injury.
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Objective:To investigate the effects of ursolic acid (UA) on proliferation, migration and iron death of ectopic endometrial stromal cells (EESCs) and its mechanism.Methods:Mouse model of endometriosis was established and the primary EESCs were isolated. The cells were treated with UA at different concentrations (0, 2.5, 5, 10, 20, 40, 50, 80, 100, 200 μmol/L). The cells were divided into Control group (normal culture), 2.5 μmol/L UA group (2.5 μmol/L UA treatment), 5.0 μmol/L UA group (5.0 μmol/L UA treatment), 10.0 μmol/L UA group (10 μmol/L UA treatment), and UA+DUSP19 group (10 μmol/L UA+50 μmol/L JAK2/STAT3 signal pathway activator DUSP19 treatment). Cell survival rate was detected by CCK-8 method. Cell proliferation was detected by plate cloning method. Transwell chamber assay was used to detect cell migration. The levels of Fe 2+ and the contents of malondialdehyde (MDA), reactive oxygen species (ROS) and superoxide dismutase (SOD) were detected by kit. Protein expression levels of Ki67, PCNA, CyclinD1, p-JAK2, p-STAT3, JAK2 and STAT3 were detected by western blot. Results:The number of clones in Control, 2.5 μmol/L UA, 5.0 μmol/L UA and 10.0 μmol/L UA groups were as follows: 152.22±15.47, 121.22±11.54, 92.00±5.54, 66.44±6.88; Ki67 protein expression was 1.08±0.10, 0.73±0.07, 0.61±0.06, 0.45±0.02, respectively; The expression of PCNA protein was 0.85±0.07, 0.64±0.05, 0.41±0.03, 0.31±0.05, respectively; CyclinD1 protein expression levels were 0.98±0.11, 0.65±0.06, 0.51±0.05, 0.42±0.07, respectively. The migration numbers were 92.78±6.27, 62.22±2.20, 50.22±4.59 and 39.11±4.33, respectively; Fe 2+ levels were (1.06±0.07) μmol/g, (1.21±0.11) μmol/g, (1.33±0.08) μmol/g, (1.47±0.09) μmol/g, respectively; MDA content was (0.48±0.06) μmol/g, (0.65±0.07) μmol/g, (0.85±0.08) μmol/g, (1.03±0.11) μmol/g, respectively; ROS contents were (19.85±1.21) %, (24.83±2.79) %, (29.04±1.86) %, (33.87±2.45) %, respectively; SOD content were (36.41±3.56) U/mg, (31.03±2.81) U/mg, (25.63±2.84) U/mg, (19.62±1.67) U/mg, respectively; p-JAK2 protein expression was 0.85±0.10, 0.75±0.06, 0.53±0.05, 0.31±0.03, respectively; p-STAT3 protein expression was 1.08±0.11, 0.79±0.06, 0.63±0.07, 0.42±0.03, respectively. The p-JAK2 protein content in UA group and UA+DUSP19 group was 0.38±0.05 and 0.75±0.08, respectively; p-STAT3 protein expression was 0.46±0.04 and 0.80±0.03, respectively; The cell survival rates were (52.55±2.44) % and (82.18±4.72) %, respectively; Fe 2+ levels were (1.57±0.06) μmol/g and (1.21±0.13) μmol/g, respectively. The differences in the above indicators between the Control group and the 2.5 μmol/L UA group, 5.0 μmol/L UA group and 10.0 μmol/L UA group were statistically significant ( P<0.05). There were statistically significant differences among 2.5 μmol/L UA group, 5.0 μmol/L UA group and 10.0 μmol/L UA group ( P<0.05). There were statistically significant differences in p-JAK2, p-STAT3, cell survival rate and Fe 2+ levels between UA group and UA+DUSP19 group ( P<0.05) . Conclusion:Ursolic acid can inhibit the proliferation and migration of EESCs cells and induce iron death by regulating JAK2/STAT3 signaling pathway, thus playing a protective role in endometriosis.
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Ferroptosis is a type of iron-dependent cell death driven by lipid peroxidation, and its mechanism is associated with iron homeostasis imbalance, lipid peroxidation, and slC7A11-GSH-GPX4 antioxidant system. Ferroptosis plays a key role in the development and progression of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), and inhibition of ferroptosis can almost completely inhibit the development of NASH. This article reviews the research advances in the mechanism of ferroptosis and its role in NAFLD/NASH and proposes the research strategies and technical means for ferroptosis, so as to provide a reference for research on the mechanism of NAFLD/NASH.